Orthopedic surgery is usually performed to repair bone injuries. It also plays a crucial role in improving the quality of life, especially in older adults. However, despite the advancement in orthopedic treatments, interactions between different factors such as pathological hormone and nutrition status, and underlying cellular and molecular mechanisms are yet to be fully understood.
Earlier animal models of orthopedic surgery included dogs, sheep, and rabbits, due to their large skeletal sizes and similarity to the human bone structure. However, these models have their shortcomings which include the husbandry cost, handling difficulties and lack of availability of transgenic animals. These disadvantages led to the popularity of rodents as the new model organism in orthopedic research. Among rats and mice, the latter has seen more popularity due to the availability of a range of genetically modified mice. Rodents, as opposed to the previously used animal models, have shorter breeding cycles and faster regeneration. These qualities combined with their lower costs and easy handling make them ideal for orthopedic research.
Before beginning the surgical procedure, it is critical to ensure that the apparatus and the equipment used are thoroughly cleaned and sterilized. Instruments that can be autoclaved must be so or disinfected using appropriate methods before use. It is also essential that the operating area be sterile and clear of any disturbances.
Before the surgery, record subject identification details such as strain and gender, and most importantly record the weight of the subject. Also, perform a physical assessment of the subject to assess its health status and activeness. Ensure that the subject has been appropriately acclimated to the facility. The acclimation process can last between a few days to a couple of weeks.
Generally, anesthesia is induced in the subject via inhalant agents. Anesthesia can be induced with the help of anesthetic systems that use a face mask or using an anesthetic chamber. The anesthetic agent amount and duration of induction are dependent on the weight of the subject among other factors. Once the anesthesia is induced, the depth of anesthesia can be verified using the toe pinch test. Additionally, physiological parameters can be monitored throughout the procedures to ensure the anesthesia is effective.
Orthopedic studies allow understanding and visualization of injuries and the effects of factors such as age and gender on the regenerative properties of the bone. Rodent models permit accurate and reproducible models of investigation that enable improvement of treatment and the quality of treatment methods in humans.
- Place the subject in a supine position on a heated mat.
- Before surgical manipulation and after induction administer analgesia. Inject bupivacaine (0.25%) proximal to the knee before surgical manipulation. Apply eye lubricant.
- Expose the surgical area by shaving the medial aspect of the hind limb. Disinfect with iodine + 70% alcohol skin scrub over 3 alternating cycles.
- Make a skin incision using scissors along the medial aspect of the hind limb proximal to the knee down to the midshaft of the tibia.
- Visually locate the diaphysis by exposing the midshaft of the tibia. Using the patella-femoral ligament as a marker, visualize the tibial plateau by flexing the knee.
- Once the patellar tendon is visible, using a rolling action of the thumb and index finger manually drill a 0.5 mm hole using a 25-gauge needle in the intramedullary canal.
- Insert 15 mm into the medullary cavity a 0.38 mm stainless steel pin until resistance is felt. Using wire cutters, cut the pin flush with the tibial plateau.
- Fracture of the tibia midshaft using straight Bonn scissors.
- Inspect the fracture site and adjacent tissues for stabilization of the fracture.
- Close with dermal staples.
Distraction Osteogenesis Protocol (Lybrand Et Al., 2015)
- Place the subject on its side with the operative extremity up.
- Make a longitudinal incision using a #15 blade scalpel from the greater trochanter to the knee in line with the femur.
- Using a micro-dissecting scissor exposes the femur by opening and splitting the muscle fascia layer between the anterior and posterior muscle compartments.
- Using a tissue elevator bluntly dissect off muscle fibers attached to the femur.
- Once the femur is visible, bluntly dissect posteriorly a path at the level of the distal femur and greater trochanter using a tissue elevator. Pass a pre-bent 18-gauge needle posteriorly around the femur for wire placement.
- Pass the wire through the needle and place it at the distal femur and greater trochanter.
- Hold the leg of the subject while applying the distraction osteogenesis device (opened 9 to 10 turns from closed position) to the femur. Ensure that before the placement the device function is validated by opening and closing it maximally.
- Thread the wires through the distraction osteogenesis device. Secure the device to the femur by grasping the ends of the wire using a needle driver and twisting it counterclockwise till an appropriate tension is achieved.
- Using a tissue elevator creates a path posterior to the femur and inserts the 18-gauge needle. Guide the second set of wires through the needle and place it at the distal femur and greater femur such that they cross the already placed wires. Tighten the wires as mentioned earlier.
- Bend the wires and cut them flush to the devices using a wire cutter.
- Using a small foot-powered circular saw generates a transverse osteotomy of the femoral shaft.
- Close the muscle fascia using a 6-0 absorbable suture followed by a 5-0 absorbable sutures for the skin around the device.
Marrow Ablation Protocol (Lybrand Et Al., 2015)
- Place the subject on its back with the operative leg maximally flexed throughout the procedure.
- Make an approximate 2 to 3 mm anterior longitudinal midline incision centered over the knee joint using #15 blade scalpel.
- Make another incision just medial to the patella and the extensor mechanism.
- Using forceps elevates and displace the extensor mechanism.
- On the visualization of the distal and the proximal end of the femur and tibia respectively, using a 27-gauge x ½ in TB syringe in the center of the proximal tibia creates an entry to the tibial medullary canal.
- Insert the syringe until resistance is felt.
In an antegrade fashion insert the stylet of a 25-gauge spinal needle through the medullary canal down to the distal growth plate. Ablate the marrow by inserting and rotating spinal needles of increasing diameter (25-gauge to 23-gauge).
- Flush the bone marrow cavity with sterile saline to remove any loose elements after the canal reaming.
- Close the incision with a 5-0 absorbable chromic gut suture.
Destabilization Of The Medial Meniscus Protocol
- Place the subject in the supine position.
- Make a skin incision from the distal patella to the proximal tibial plateau.
- Make an incision using scissors in the joint capsule immediately medial to the patellar tendon and spread open.
- Retract the patellar tendon and expose the cranial meniscotibial ligament of the medial meniscus by blunt dissection of the fat pad.
- Section the meniscotibial ligament of the medial meniscus using a micro-surgical knife or #11 scalpel.
- Using 7-0 Vicryl close the wound cutaneously.
Calvarial Defect Protocol (Spicer Et Al., 2012)
- Induce anesthesia in the subject and shave the fur from the bridge of the snout between the eyes to the caudal end of the skull/calvarium.
- Make an approximately 1.5 cm incision down to the periosteum over the scalp from the nasal bone to just caudal to the middle sagittal crest or bregma using a scalpel.
- Apply lateral counter traction and visualize the calvarium. Using a scalpel, sharply divide the periosteum and gently push it laterally while elevating from the skull using the elevator.
- Retract to spread soft tissues and expose the underlying bone. Irrigate with sterile normal saline.
- Using a surgical drill and trephine operating at ≤1500 rpm, score the calvarium. Irrigate the with sterile normal saline dropwise at approximately 1 drop every 2 seconds.
- Continue trephination and apply very gentle pressure while processing the trephine around the scored defect margins.
- Place into and move the elevator blade circumferentially around the defect margin. Gently lift with the elevator while applying gentle pressure to complete the defect. Slide the elevator blade under the freed calvarium and free the dura from the underside of the bone by sweeping back and forth with the blade.
- Using the elevator raises the calvarium off of the dura. Carefully remove ant bone fragments using the elevator.
- Copiously wash the defect with sterile normal saline to remove any debris/bone chips. Place the implant material into the defect.
- Using 4-0 monocryl suture close the periosteum over the implant.
- Using a running or simple interrupted 3-0 plain gut suture close the skin over the periosteum.
Total Hip Replacement Arthroplasty (Powers Et Al., 1995)
- Make a subcutaneous incision from the mid femur to the dorsal midline.
- Expose the dorsal and anterior muscles of the lateral hip by making an incision through the cutaneous Maximus
- Sever the gluteal muscles attachments from the greater trochanter of the femur.
- Open the joint capsule and expose the femoral head. Cut the ligament of the femoral head and luxate the head.
- Excise the femoral head and ream the intramedullary cavity using a 3/64-inch drill bit to a depth of 15 mm. Follow this by reaming the cavity using a No. 40 dental reamer.
- Using an 18-gauge needle, insert approximately 1 ml of PMMA. Place the femoral component exposing 1.5 mm of the prosthesis above the level of resected bone.
- Elevate the gluteal musculature from the dorsolateral surface of the iliac wing. Flatten the anterior rim of the acetabulum and ream the acetabulum using a carbide 6 mm, oval denture burr.
- Fix the acetabular component to the pelvis using 2 polymeric screws passed between the tables of the pelvis.
- Reduce the hip and place a 3-0 Dexon suture starting at the posterior aspect of the proximal femur, passing next to the bone, and continuing medial to the acetabular component and adjust it to the femur on the anterior lateral surface.
- Using a 4-0 Dexon suture the gluteal muscles, cutaneous Maximus muscle, subcutaneous tissue, and skin.
Standard Closed Femoral Fracture Protocol (Bonnarens And Einhorn, 1984)
- Completely flex the knee and make a 1 cm incision just medial to the patella.
- Divide medially, the longitudinal fibers of the quadriceps mechanisms and dislocate the patella laterally to expose the femoral condyles.
- Ream the canal using a 20-gauge needle inserted between the condyles. Introduce a 0.45 mm Steinmann pin into the canal and drive it in retrograde fashion up the shaft exiting through the greater trochanter.
- Cut the pin flush with the cortex of the patellofemoral groove and bury it beneath the bone.
- Make an incision over the greater trochanter to access the proximal end of the pin. Create a 90-degree bend in the pin and cut it leaving a 3 mm handle. Close the wound.
- Place the subject in a supine position. Place the pinned femur in abduction and external rotation and position it over the center of the animal support stage.
- Raise the support stage until the blunt guillotine blade is snug with the thigh.
- Drop the weight from a height to fracture the femoral diaphysis.
Post-Operative Care And Pain Management
Pre-emptive measures to avoid postoperative pain in the subject can be done by administering a dose of opioids such as buprenorphine before incision. After the surgery, the subject must be kept warm in a recovery unit using hot water blankets, hot water bottle, heat pads, and warm sterile saline should also be administered to the subject before returning it to its home cage.
Recovery from anesthesia should be monitored closely and respiratory support, if needed, should be provided. Analgesic should be maintained postoperatively for up to 48 hours in increments of 24 hours or as required. The use of NSAIDs should be avoided as they tend to interfere with the bone healing process. The subject can be returned to its home cage once it has recovered.
Although rare, post-operative prophylactic antibiotics can be administered to prevent infections. Look out for signs of infections such as swelling, lethargy, and purulent drainage. Infection can affect bone healing, thus if an infection is noticed the subject should be euthanized.
Surgery-specific complications and issues may occur post-operatively. Wound dehiscence should be dealt with by re-suturing the wound under anesthesia. If repeated re-suturing fails, allow the wound to heal by secondary intention using an antibiotic ointment. Surgery involving pin placement may have an occurrence of pin slippage. Pin slippage may be visualized outside the skin. Remove the pin using needle driver. In marrow ablation protocol, iatrogenic tibia fracture risk exists while instrumenting the medullary canal. Tibia fracture may be indicated by the subject’s inability to bear its weight on the operated extremity or by the presence of deformity. In such a situation the subject must be euthanized.
Micro-computed tomography revealed a transient reduction in fracture size by either CBD or THC four weeks after the fracture which reached control level after weeks 6 and 8. Further, infrared spectroscopy confirmed the involvement of CBD in increasing the collagen crosslink ratio, which is linked with improving the biomechanical properties of the fracture callus. (Kogan et al., 2015)
Evaluation of the effects of Nrf2 deficiency in fracture healing
The investigation was conducted using nuclear factor erythroid 2-related factor 2 knockout (Nrf2-KO) mice that were subjected to standard close femoral shaft fracture. Wild-type mice that had also undergone femoral fracture were used as a control group for the investigation. Results from the investigation revealed that Nrf2 expression is activated during fracture healing. Analysis data suggested that Nrf2-KO mice developed significantly fewer callus tissues and showed delayed bone healing and re-modeling in comparison to the wild-type control group. From the investigation, it was concluded that Nrf2 played a crucial role in bone regeneration. Thus, it was suggested that pharmacological activation of Nrf2 could have a potential therapeutic effect in fracture healing. (Lippross et al., 2014)
Evaluation of cisplatin on bone healing during distraction osteogenesis
Stine et al. studied the effects of cisplatin (CDP) on bone regeneration during distraction osteogenesis (DO) in C57BL/6 (B6) and tumor necrosis factor receptor 1 knockout (TNFR1KO) mice. B6 mice were evaluated under two regiments of dosages; CDP exposure during early stages of DO and CDP treatment on DO. CDP-treated mice had a significant decrease in the mineralized area of distraction gap compared to vehicle-treated mice as observed in the distracted tibial radiographs. Further, the histological analysis confirmed a significant decrease in the cellular bone formation of the DO gaps of CDP-treated mice. For the second regiment of CDP doses, mice were treated with the same dose of CDP for two consecutive days followed by DO one week later. A comparison of distracted tibial radiographs showed no difference between CDP treated and vehicle-treated mice. On the other hand, neither CDP nor vehicle treated TNFR-1 deficient mice showed a prominent decrease in the mineralized area in the distraction gaps. However, a statistically significant difference between CDP treated mice and vehicle-treated mice were observed in the decrease in cellular bone formation. The study concluded a profound short-term negative effect of CDP in DO bone repair process.
Evaluation of the effect of FGFR3 on fracture repair
Xie et al. evaluated the role of FGFR3 in fracture healing using Fgfr3 knock out (Fgfr3-/-) mice that were maintained on C3H/HeJ background. The mice underwent tibial shaft fracture, and analysis was performed to investigate the effect of the deficiency on the healing processes of the fracture. X-ray radiographs and micro-CT analysis showed that in comparison to the control wild-types, Fgfr3-KO mice showed accelerated formation and remodeling of the fracture callus. Regarding biomechanical properties examined using the three-point-bending test at weeks 3 and 4 post-fracture, the knockout mice showed greater stiffness at day 21. However, no significant difference could be observed between the knockout and control wild types on day 28. Although an increase in the ultimate force was observed in the Fgfr3-KO, work to failure required was comparable between both the groups at days 21 and 28 postfracture induction. The investigation suggested that down-regulation of FGFR3 activity could be a potential bio-therapeutic fracture treatment based on the observation of accelerated fracture callus mineralization and up-regulated expression of osteoblastogenic genes in Fgfr3-/- mice
Evaluation of the effect of local vibration and pulsed electromagnetic field on bone fracture
Male Sprague-Dawley rats that were subjected to tibial osteotomy were utilized to investigate the possible therapeutic effect of local low-magnitude, high-frequency vibrations (LMHFV) and pulsed electromagnetic field (PEMF) on the bone healing process. A mechanical stimulator was used to conduct vibrations for 15 min/day using the clamp method in the LMHFV group to overcome the limitations of whole-body vibrations. This method allowed control of vibration magnitude and frequency while exposing the tibia to the vibration in a fixed position. For the second group, PEMF treatment was performed each day by restraining the subject and delivering the PEMF generated by two pairs of Helmholtz coils surrounded by a Faraday cage. Both treatments were started 5 days post-operatively. Analysis of the radiographs taken 21 days after the end of the healing process, although not significant, showed enhanced callus formation, obliteration of the fracture line and bridging of the fracture gap in the treatment group as compared to the control group. The stereological analysis showed a significant difference in the summed area of the new bone area between all three groups. Further, LMHFV treatment was observed to have preserved more trabecula as opposed to the control group. Statistically, a significant difference was also observed between the three groups in terms of cartilage summed area. Based on the observations made during the investigations it was suggested that osteoblasts are sensitive to low-magnitude, high-frequency vibrations. (Bilgin et al., 2017)
Investigation of bone fracture healing in splenectomized rats
Splenectomy is required in fracture patients with blunt abdominal trauma and failure of conservative management. Xiao et al. investigated the effect that splenectomy has on the bone healing process using male Sprague-Dawley rats that underwent femoral fracture. The subjects were then divided into two groups. One of the group followed the fracture with splenectomy (fracture + splenectomy) while the other group only underwent spleen isolation (fracture group). Splenectomy was performed by making a vertical incision at 1.5 cm under the left costal margin and isolating the spleen using blunt forceps. Splenectomy was followed by ligation of blood vessels and subsequent removal of the spleen. The abdominal wall and skin incision were then sutured. Results from the investigation suggested splenectomy inhibited recruitment of macrophages and the production of inflammatory cytokines. Further, fracture healing was delayed in the splenectomy group as evident from the histological analysis.
Investigation of conjugated linoleic acid in promoting fracture healing
Inspired by the clinical observation of fracture healing in Tibetans, Shan et al. aimed to investigate fracture healing capabilities of conjugated linoleic acid (CLA), which is a prominent part of Tibetan daily diet. In their investigation, they made use of Sprague-Dawley rats that were maintained on either a basal only diet or CLA with basal diet. The rats were subjected to standard tibial fracture procedure. CLA effect was quantified using combined structural evaluation (X-ray and micro-computed tomography), biomechanical test, and histological examination. Radiological evaluation of the fracture healing process was assessed on weeks 2, 4 and 6 and degree of healing was using a 0 to 5 point system with 0 being no callus and 5 being callus resorption after the solid union. Biomechanical test and histological analysis of the tibial bone was performed at week 6. At week 6 a radiological evaluation score of 4.4 ± 0.6 was recorded for the CLA group; 40% of the cohort had complete callus absorption. In contrast, the control group obtained a score of 3.4 ± 0.5. Further, micro-CT analysis at week 6 revealed CLA group to have significantly higher values for bone mineral density, bone strength index and cross-sectional area of the callus. Load to failure values of the CLA group (78.12 ± 10.03 N) as determined by three-point bending test were also statistically significant than the control group. The investigation of Shan et al. showed that CLA improved the quality and mechanical strength of fracture healing in rat callus, thus suggesting the potential therapeutic applications CLA in fracture healing in patients.
Evaluation of whole-body vibrations in improving fracture healing in ovariectomy-induced osteoporosis rats
Osteoporosis leads to an imbalance in the bone tissue absorption and replacement resulting in weaker bones that are prone to fractures. This condition is often accelerated by age and commonly seen in post-menopausal women. In their investigation, Butezloff et al. investigated the effect of whole-body vibrations on fracture healing in rats that underwent ovariectomy. For their experiment, Butezloff et al. used 7-week old female Wistar rats that were divided into ovariectomy and sham groups. Ovariectomy was performed by bilateral extraction of the ovaries through a dorsolateral approach. Sham models only had their ovaries exposed but otherwise left undisturbed. Both groups underwent a closed fracture procedure at mid-femur three months after ovariectomy or sham surgery. Three days following the fracture procedure, rats were subjected to a whole-body vibration therapy by placing them in a special acrylic cage on a vibrating platform (peak-to-peak displacement of 1 mm at a frequency of 60 Hz). Both the ovariectomy and sham group received 20 minutes whole-body vibration therapy 3 days every week over the course of 14 or 28 days. Data from the investigation revealed that ovariectomized rats had significantly lower bone density and bone content in comparison to the controls. However, it was also observed that whole-body vibration therapy partially protected against bone loss in the ovariectomized rats though not in the controls. Data analysis from the investigation suggested that vibration therapy led to the improvement of the quality of the bone and fracture bone callus in ovariectomized rats.
Investigation of the effect of TLR4 activation on the healing of calvarial defects
Wang et al. tested their hypothesis that Toll-like receptor 4 (TLR4) activation plays a role in the healing of calvarial defect using wild-type C57BL-6J and TLR4 knockout mice. Both, wild-type and TLR4 knockout mice underwent procedures to create a 1.8 mm diameter calvarial defect. Analysis of bone healing was performed over the course of 28 days by the end of which both groups had achieved bone remodeling. By postoperative day 21 both wild-type and the TLR4 knockout group showed comparable healing. However, calvarial healing was accelerated in TLR4 knockout mice as observed from the radiographic and histomorphometric analyses. Based on the observations and analyses, the study provides evidence to suggest TLR4 knockout mice showed accelerated bone healing with earlier and higher expression of inflammatory cytokines and increased osteoclastic activity.
Evaluation of focused low-intensity pulsed ultrasound in enhancing bone regeneration
The effect of low-intensity pulsed ultrasound (LIPUS) on the induction of bone repair is a well-researched subject. With their investigation, Jung et al. aimed to evaluate the bone regeneration potential of focused low-intensity pulsed ultrasound when selectively applied to the fractured calvarial bone. Eight-week-old male Sprague-Dawley rats underwent creation of a 4 mm calvarial bone defect and divided into two treatment groups that either received or did not receive focused LIPUS treatment. Focused LIPUS set up consisted of a spherical segment focused ultrasound transducer that operated at a frequency of 650 KHz. Sonication pulses were created using a function generator that was amplified using a frequency power amplifier to drive the transducer. The treatment group received 20 minutes of focused LIPUS twice a week for 8 weeks. The control group also received the same treatment, except with the focused LIPUS machine turned off. At 8-week post-treatment, CT image analysis showed that treated group 2.2-fold higher regeneration of the defect area as compared to the controls. Similarly, a 1.9-fold higher calcein emissions were observed in the treated group in the defect area than in controls. Histological analysis, post-exposure also showed focused LIPUS treated group had a relatively significant increase in proliferating cells as compared to the controls. The study concluded that focused LIPUS improved re-ossification through cell proliferation enhancement in the calvarial defect sites.
Advantages And Disadvantages Of Rodent Models
In comparison to large animal models such as dogs and sheep, rodent models offer many advantages. Despite their large size being an advantage, the handling and maintenance of large animals are difficult. Additionally, the cost of husbandry is high in comparison to that of rodents. Large animal models also lack in availability of transgenic animals. Rodents, on the other hand, are economical since they are inexpensive and have shorter breeding cycles. Further, rodents are well-researched animals, and much is already documented with respect to their biological processes and responses to diet modifications and drug administration. The availability of athymic, transgenic and knock-out rodents also makes them a viable choice for different investigations.
However, rodent skeletal system does have a significant distinction from the human skeleton. Unlike human skeleton, rodent skeleton continues to grow and reshape throughout their life cycle. Growth plates in rodents remain open well into their adulthood. With the advancement in age rodents show loss of cancellous bone, thinning of cortical bone, and increased cortical porosity as seen in humans. Their reduced lifespan makes them ideal for studies investigating the effects of aging in bone metabolism and regeneration processes. Rodents so require working within their biological constraints and their small size may also not be suitable for modeling of certain orthopedic investigations. Their use in chondral defect repair investigations is limited due to the thinness of their cartilage layer. Rodents also vary significantly from humans in their gait pattern and biomechanical loading environment.
Orthopedic research involves the improvement of treatments of musculoskeletal system conditions.
Rodent models are preferred over large animal models due to their low maintenance and cost, shorter breeding cycles and faster regeneration.
Availability of athymic, transgenic and knock-out rodents makes them ideal for various research requirements.
The reduced lifespan of rodents allows age-dependent investigation.
Rodents show loss of cancellous bone, thinning of cortical bone, and increased cortical porosity with age as seen in humans.
Rodent strain, age, and weight among other factors an influence orthopedic investigations.
Anesthesia induction can be done using inhalant or injectable agents. The depth of anesthesia should be verified before beginning surgical procedures.
During surgical procedures, care must be taken not to damage surrounding tissues or bones.
A recovery area should be set-up and fluids should be replaced by subcutaneous or intraperitoneal injection of warm sterile saline.
Infections may influence the results of the investigation, hence, euthanizing the subject is recommended should it occur.
Appropriate pain management techniques should be followed.
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