Development of a Novel Internal Fixation Model for Rat Radial Fractures: Fracture Healing Assessment and Dorsal Root Ganglion Isolation

Radial fractures are highly prevalent globally and a leading cause of extremity long bone fractures. The rat radial fracture model is crucial for studying fracture healing, drug screening, and biomaterial evaluation in orthopedic research. Since the radius of rats it's relatively thin, the steel plates and screws currently used in clinical practice are not suitable for the treatment of rat radial fractures.

To overcome this limitation, the protocol innovatively combines suture and titanium needle fixation, establishing a stable radial fracture model for internal fixation research. In the protocol, we also presented the use of the live animal micro-CT imaging system to clarify the fracture healing in rats. We also showed the extraction and culture of the DRG in the rat fracture model, which will be used to study the mechanism of nerve injury and repair in fractures.

The rat fracture surgical instruments include scalpel blade, scalpel handle, operating scissors, needle holder, hemostatic forceps, and toothed tissue forceps. The male Sprague Dawley rat, approximately 8 weeks of age and weighing about 200 grams. Anesthetize the rat using an isoflurane gas anesthesia machine and maintain ventilatory anesthesia throughout the surgical procedure.

Maintain the operating room temperature at 28 C.Place the rat in the lateral position. Expose the right forelimb, and shave off the hair using a hair clipper. Drape sterile sheet and wrap a tourniquet around the proximal right forelimb to reduce surgical bleeding.

Prepare the skin by alternating alcohol and betadine scrubs three times. Make a 1.5 cm longitudinal incision on the dorsal radial side of the right forearm. Separate the muscle spaces and fully expose the right forelimb radius.

Use operating scissors to cut the radius to create a transverse fracture. The direction of the scissors forms a 90-degree angle with the radius. Take a 0.8 mm diameter, 10 mm long titanium needle, and insert it into the radial fracture along its longitudinal axis.

Fix the radius and the titanium needle with suture. The suture was tied with three consecutive knots, and the titanium needle was secured to the radius via four interrupted sutures. Suture the skin color of the excess suture and close the wood.

Disinfect the skin with betadine scrubs. Cover the wound with sterile gauze and secure it with bandages. The rat regained consciousness after anesthesia and its condition recovered well.

Anesthetize the rat using an isoflurane gas anesthesia machine. Place the rat in a prone position within the scanning chamber. Adjust limb and head positioning to center the target scanning area in the field of view and maintain continuous ventilation and anesthesia throughout the procedure.

Push the fixed animal bed into the scanning chamber and close the chamber door. The micro-CT scanning parameters were set as follows:scan mode:standard, exposure time:two minutes, field of view:72 mm, voxel size:144 m, kilovolt:90;micro-amperes:88, filter:3D-soft. The scan data is automatically transmitted to the software, and three dimensional CT image is generated.

Separate the target tissues. Export the processed images. Sever the spine, remove the C1 to C7 vertebral bodies.

Cut the cervical spine along the anterior and posterior midlines, divided into left and right halves. Use toothed tissues forceps to pick up the DRG, gently lift it up and then use operating scissors to cut the nerve fibers. Transfer extracted DRGs to a 1.5 mL centrifuge tube with extraction medium.

Place on ice. Slowly aspirate and discard the extraction medium. Add 1 mL of mixed digestive and enzyme working solution.

Digest in a 37 C water bath for 60 to 70 minutes. Pipette an appropriate amount of the fibronectin working solution into a Petri dish and coat it in a 37 C incubator for an hour. Post digestion, aspirate, and discard the mixed digestive enzyme working solution.

Collect filtrate, centrifuge at 70 x g for 8.5 minutes. After centrifugation, aspirate and discard the supernatant in the centrifuge tube. Add 1 mL of complete medium to resuspend the precipitate.

Take out the Petri dish. Discard the fibronectin working solution and wash with PBS twice. Inoculate the cells into a Petri dish, add 1 mL of complete medium.

Place them in a constant temperature incubator at 37 C with 5%carbon dioxide for 8-12 hours. The micro-CT imaging confirmed the successful establishment of radial fracture rat models and internal fixation model for rat radial fractures. 20 days post modeling, micro-CT analysis of SD rats in the fracture model group revealed favorable prognosis.

Callus formation was observed bridging the fracture ends, with progressive fusion and gradual obliteration of the fracture line, which is consistent with the early phase of bone union. Images showed a blurred fracture line in the modeled region, suggesting that bone tissue at the fracture site had largely healed. On the 5th to 6th day after DRG isolation, neuronal cells exhibit extensive cytoplasmic exposure.

At this stage, the entire neuronal cell bodies and their surrounding synapses are clearly distinguishable. Immunofluorescence microscopy was used to photograph neuronal cells on the 6th day of culture. As shown in Figure 9, Beta III-tubulin was shown as green fluorescence.

Beta III-tubulin positive cells including both DRG cell bodies and axons were stained green, and the blue fluorescence represents the nuclei of neurons stained by DAPI. Rats are a classical model for studying the mechanism of fracture healing due to their clear genetic background and short reproductive cycle. However, the small radius diameter about 1-1.5 mm makes it impossible to match clinical-grade internal fixation device, severely restricting its utility in nerve-bone healing research.

In response to this problem, this study innovatively proposed the combined suture titanium needle internal fixation method. The core logic is to replace the rigid fixation of screws with the flexible ligation of sutures, which could reduce the iatrogenic nerve injury and provide stable mechanical environment. This protocol provided repeatability and standardization for the construction of the model.

The sutures and titanium needles are used for internal fixation in rat radial fracture models. Fracture healing is assessed via micro-CT. The primary DRG neurons are isolated and cultured following fracture healing examinations.

In the protocol, we innovatively combine sutures and titanium needles for stable internal fixation in rat radial fracture model. Fracture healing was assessed via micro-CT, and primary DRG neurons were isolated and cultured for further investigation.