Abstract
Background/Aim: As one of the common clinical diseases, fractures have many causes, mechanisms, healing and influencing factors; especially fracture healing is a long-term and complex process. Animal fracture models can simulate the various states of human fractures, and on this basis, the prevention, mechanism, and treatment of fractures can be studied to further guide clinical practice. Materials and Methods: Here, we developed a novel and portable device to create a closed fracture model in mice. We then compared this novel closed fracture model with the traditional open model in multiple dimensions to evaluate the modelling process of establishment and healing. The two models were evaluated by imaging, immunostaining, and behavioral tests, which fully demonstrated the stability, universality and operability of the modified fracture model in mice. Results: Surgical quality assessment revealed that the closed fracture model had a shorter operation time and smaller wound than the open model. X-ray and micro-CT results showed no differences between the two models in the evaluation of radiographic and morphological changes during fracture healing. Histological examination revealed the process of the typical intrachondral osteogenic pathway after fracture. Moreover, animal gait analysis indicated reduced postoperative pain in the closed group compared to the open group. Conclusion: This study provides a constructive strategy for a closed fracture model in mice and demonstrates the effectiveness and feasibility of the closed fracture model in studying the typical intrachondral osteogenic pathway of fractures from multiple dimensions.
Fractures are the focus of orthopedic trauma epidemiology and are associated with a substantial burden on global health systems. This burden increases with the aging of population and traffic accidents (1). Globally, there were 178 million [95% uncertainty intervals (UI)=162-196] new fractures in 2019, marking an increase of 34% (30-13.7) since 1990. Additionally, there were 455 million (428-484) prevalent cases of acute or long-term symptoms of a fracture [a 70.1% (67.5-72.5) increase since 1990] and 25.8 millions (17.8-35.8) years lived with disability [a 65.3% (62.4-68.0) increase since 1990] (2). These statistics negatively impact the health and quality of life of patients, placing a significant strain on families and society as a whole. Most fracture patients can be cured after clinical treatment but affected by factors, such as postoperative infection and poor bone healing ability of patients, the healing effect of 5% to 10% of patients with bone nonunion is very poor, and there is a risk of amputation. Therefore, it is more urgent to conduct fracture research and analyze the factors that promote fracture healing, which is very important for fracture prevention and prehospital emergency treatment.
Animal fracture models, designed to develop new bone repair treatment strategies, have been widely used in preclinical studies to understand fracture healing mechanisms and as proof-of-concept, greatly benefiting human disease research and biomedical development (3). Due to the many problems of large animal models, such as the long time needed for the bone healing process in captivity, the difficulty in developing gene knockout animals and the need for a large amount of research funds, smaller animal models are increasingly recognized and widely used, such as mouse models (4-6). Because of its low price, short experimental period, repeatable and standardized growth environment, detailed genome map, and many other advantages, mice have great potential in constructing various types of fracture models to explore the mechanism of fracture healing. Currently, the most commonly used methods for constructing fracture models in mice involve open fracture and closed fracture models.
The ideal experimental fracture should be stable, repeatable, and convenient concerning the site, degree of displacement, rigidity of fixation, and soft-tissue injury (7). The femur is the most prominent tubular bone in both the human and mouse bodies, consisting of the femoral shaft and metaphases at both ends (8). Femur fracture is one of the most common types of lower limb fracture. These are easily reproduced in animal models, leading to frequent use in open fracture models of mouse femurs (9-11). Due to the small size of the femur, femur fractures are less likely to occur in animal models, especially mice, while the open surgery method fully exposes the femur and soft tissue, creating a simple and cost-effective model. Therefore, it is a more common surgical method and an effective fracture method. However, open exposure of the bone induces surgical trauma, including retraction of the surrounding muscles and damage to the extraosseous vasculature. Another model, the closed fracture model, more closely mimics clinical situations of bone fracture and can be induced by the 3-point bending device or a blunt guillotine device without surgical exposure of the bone. However, these devices are not only large and hard to move or tear down, but also expensive to use and complicated to operate. Thus, we need a lightweight and portable device to construct a closed fracture in mice.
Constructing a stable and reliable mouse femur closed fracture model with a delicate surgical instrument is necessary to investigate the pathogenesis and molecular mechanisms of bone tissue injury, remodeling, and healing. In this study, we designed and implemented a novel, portable, and efficient femur fracture device and compared two mouse femur fracture models using this novel non-invasive fracture instrument and an open-operative method to learn the establishment and healing processes of different fracture models. Meanwhile, we thoroughly explained the surgical procedures and operative points of these two types of femur fracture models to ensure repeatability and stability. Additionally, we employed imaging, immunostaining, and behavioral evaluations to further assess these models, providing a reference for mouse fracture models in learning the process of establishment and healing.
Materials and Methods
Study design and mice. The objective of this study was to establish two mouse models of femur fracture and compare the operational feasibility of these models. Figure legends list the surgical protocols and the number of replicates for each experiment. C57BL/6 WT mice (4-week-old) in this study were purchased from Xiamen University Laboratory Animal Center. For this study, we only used male C57BL/6 WT mice because sex has an impact on hormones and cell-mediated immune response in mice (12, 13). All mice were housed in the Laboratory Animal Centre at Xiamen University under usual pathogen-free conditions. All animal experiments were approved by the Ethical Review Committee of Xiamen University (Xmulac20190084).
Establishment of the open femur fracture model in mice. Before the operation, each mouse was examined for healthy physical condition, free of skin ulcerations or hair loss. Prior to the procedure, erythromycin ointment was applied to both eyes. All surgical instruments were thoroughly sterilized. The detailed step-by-step protocol for surgical femur fracture is as follows:
1. The hair from the right iliac crest to the right ankle was completely shaved. The skin was cleansed with iodophor disinfectant three times to disinfect the area and remove loose hairs from the surgical site. The skin area of the knee joint requires intensive disinfection (Figure 1B, i).
2. Under general anesthesia (2.5% isoflurane in oxygen) with a rhinophore, the mouse was placed on a warming pad in the left lateral position. After palpating the femur, a medial 0.5-cm skin incision was made longitudinally along the thigh to expose the muscle. Large tendons attached above the greater trochanter of the femur can serve as an anatomic marker for muscle stripping to expose the femur (Figure 1B, ii).
3. The sterile needle was inserted into the intercondylar fossa and retrograded into the bone marrow cavity longitudinally along the femur, stopping immediately upon feeling an empty sensation. The needle was gently pushed into the bone marrow cavity with the operator’s fingers during needle insertion. The needle should not be rotated to prevent loosening the hole and causing the fixing needle to fall off easily. During this step, the operator should not puncture the cortical bone, nerves, or blood vessels with the needle (Figure 1B, iii). Different types of needle fixation are required for femur fracture operations in mice of different ages (Figure 2A-B). The anteroposterior and lateral positions of the mice were detected by X-ray after the needle was inserted into the bone marrow cavity longitudinally along the femur.
4. Blunt dissection of the mid-femur without injury to the periosteum is recommended to fully expose the femur (Figure 1B, iv). A special oral probe was inserted below the femur to facilitate further mechanical osteotomy (Figure 2C).
5. The middle of the femur shaft was cut transversely by the dental probe using a handheld saw on a low-power setting (Figure 2D). After completely disconnecting the femur, the proximal femur is held with tweezers. The fixation pin located in the distal femoral bone marrow is threaded longitudinally along the femur to the proximal end of the bone. The needle should not be rotated to prevent loosening the hole and causing the fixing needle to fall off easily. Adjusting the angle and depth of the fixation pin to achieve anatomic reduction is crucial to the success of the operation. Clamping the femur tightly during the procedure may damage the periosteum (Figure 1B, iv). Finally, the needle was cut as flush to the cartilage as possible in the intercondylar notch, avoiding the weight-bearing area of the joint.
6. The incision was rinsed with a physiological saline solution. Then, the muscle, subcutaneous fascia, and skin were sutured layer by layer (Vicryl Rapide 5-0 sutures; Ethicon, Somerville, NJ, USA) and disinfected with iodophor (Figure 1B, v). The mice remained on the warming pad until they recovered from anesthesia. They were then placed back in their cages and given access to food. Each mouse received 40,000 U/day of penicillin injections for three days following surgery to prevent infection and 0.5 mg/kg daily of tramadol hydrochloride subcutaneous injections for analgesia.
The design of a novel femur fracture device. (A) The closed femur fracture device comprised hemostatic forceps (14 cm) and a U-shaped metal groove to construct the 3-point bending device. (B) The key steps of constructing the open femur fracture model in mice. Schematic representation of the surgical operation for the open femur fracture model in the upper left corner of the figure. (C) The key steps of constructing the closed femur fracture model in mice. Schematic representation of the operation for the closed femur fracture model is shown in the upper left corner of the figure.
(A) Different sizes of syringe needles used for mice of different ages. (B) Detailed instructions and recommendations for different sizes of syringe needles used for intramedullary needles. A dental probe (C) and a dental wire cutter (D) were used to construct an open fracture model.
Establishment of the closed femur fracture model in mice based on a novel instrument.
1. This step is consistent with step 1 of the open fracture model (Figure 1C, i).
2. After general anesthesia, following step 3 of the open fracture model, the internal fixation pin was propelled longitudinally along the femur to the proximal end of the femur (Figure 1C, ii). The retrograde insertion of the needle into the femoral canal was done through the skin without exposing the intercondylar notch of the kneed.
3. After palpating the femur, the novel closed fracture instrument was applied perpendicularly to the longitudinal axis of the femur (Figure 1C, iii). The femur was clamped firmly, and the process was stopped when the break was felt or the cracking sound of the bone was heard (Figure 1C, iv).
4. Finally, the needle was cut, and the knee was cleansed with iodophor disinfectant for disinfection without suturing (Figure 1C, v). The postoperative medication was consistent with the procedure of the open fracture model.
Surgical quality assessment. The first part is the evaluation of surgical quality, and depends on the operative time, amount of bleeding and the number of surgical instruments. The second part is the comprehensive quality evaluation after operation, depending on the clinical outcome, the incidence of complication (claudication), and the incidence of wound infection. The mouse gait analyzer used for the assessment of claudication is presented in Table I.
Surgical quality assessment.
Radiographic assessments. A micro-X ray imaging system (Aolong, Liao Ning, PR China) was used to observe fracture healing with the following parameters: tube voltage=60 kv, tube target flow=0.6 mA. Radiographs at different post-fracture time points were evaluated blindly by three independent investigators using a radiographic-scoring scale system based on the bone bridge of the cortices and the bone callus of healing presented in Table II. Modified RUST scoring is an altered RUST score model that requires investigators to evaluate the presence of a callus, bridging, or remodeling rather than only if it is present or absent. The modified RUST score has a value from 4 to 12 and is shown in Table III.
Radiographic-scoring scale system.
Modified RUST (RUSF) system.
Computed tomography-scan. After the animals were euthanized, the femur specimens of the mice fixed in 4% paraformaldehyde were analyzed at a resolution of 10 μm on a high-resolution micro-CT Scanner (Skyscan 1,272; Skyscan, Aartselaar, Belgium). Three-dimensional structure and morphometry were performed with the nrecon software (NRecon 1.0, Skyscan), and morphometric parameters defining microarchitecture, including bone volume/tissue volume (BV/TV) and bone volume (BV), were performed by CTan software. The femur samples after uCT scanning were placed in 5% EDTA for the follow-up experiments.
Histological staining analyses (HE and SO&FG). The bone tissues were fixed with 4% paraformaldehyde at 4°C overnight. Decalcified samples were embedded in paraffin and sectioned at 5 μm intervals. 0.1% Safranin O/0.5% Fast Green (SO&FG) was used to stain the femur sections, in which the increase of cartilage during remodeling of the bone fracture was evaluated. Leica’s TCS SP8 DLS confocal microscope was used to image immunofluorescence sections, and Leica Application Suite X software was used to quantify the fluorescence signals (Leica, Germany). The sections were imaged via a microscopic digital section scanning system (Motic VM1, MOTIC, Xiaman, China) and DSS canner software (MOTIC).
RNA Isolation and qRT-PCR. RNA samples extracted from bone (RNeasy Plus Micro Kit, Qiagen, Hilden, Germany) were qualified and quantitated by NanoDrop (ThermoFisher Scientific, Danvers, MA, USA). High-Capacity cDNA Reverse Transcription Kits (Vazyme, RL201-01) were used to reverse transcribe mRNA and to synthesize cDNA. qRT-PCR was conducted using a ChamQ SYBR Color qPCR Master Mix (Vazyme, Nan Jing, PR China), and the following primers: Col2a1 (NM_031163.4), Col1a1 (XM_021213774.2). After adjusting for the expression of the positive control, the relative expression levels of mRNA in each sample were computed and quantified using the 2−∆∆Ct method. Hprt was used as internal control for normalization.
Protein extraction and western blot analysis. The bone tissue samples were frozen and grinded with liquid nitrogen. Non-specific signals were blocked with 5% skimmed milk for 1 h and the membrane was incubated overnight at 4°C with specific primary antibodies: OPN (Cell Signaling, #27927, 1:2,000), OCN (Abcam, ab93876, 1:1,000), and GAPDH (Cell Signaling, #51332 1:1,000). Immunoreactive bands were detected using the Image Quantla-4000 imaging system (Gel Doc XR+, BIO-RAD, Hercules, CA, USA) and the protein signals were quantified by the Image J software (1.8.0_172).
Gait analysis system. Mice used for gait analysis needed to be familiar with the instrument environment and were trained before gait analysis to ensure the accuracy of the data. Data were collected from the mice, and real-time natural gait behavior videos were made for each group 3 times, via the gait imaging analysis system for rodents (MSI DigiGaitTM, USA). The qualified videos contained 7~8 consecutive strides. After that, the special analysis software DigiGait (Mouse Specifics, USA) was used for analysis and the mean value of each indicator was taken. Relevant indicators are paw area (cm), the percentage of stance stride (%), the stride length (cm) and sciatic function index (SFI).
Statistical analysis. All data are presented as the mean±SD. After testing for homogeneity of variances, unpaired Student’s t-test (two-tailed) and two-way ANOVA with Bonferroni correction were performed in experiments as indicated in the figure legend. The default alpha and beta values were set to 0.05 and 0.8, respectively. The GraphPad PRISM software (v6.01, La Jolla, CA, USA) was used for statistical analysis. A p-Value of 0.05 or less was considered as statistically significant.
Results
The design of a novel femur fracture device. This closed femur fracture device was based on a modified hemostatic forceps (14 cm) derived from daily surgical and experimental experience (Figure 1A, i). A suitably sized metal groove was welded to one of the top ends of a hemostatic forceps. The other top end was ground to fit the metal groove (Figure 1A, ii-iii). The hollow of the metal groove can be caught vertically in the middle of a mouse’s thigh, causing a femur fracture with the right amount of force. Notably, the top ends of the forceps are attached to the medial and lateral femur rather than the anterior and posterior, to avoid sciatic nerve injury. The fracture lines of the mice incorporated into this study were all in the middle of the femoral shaft with the help of the novel femur fracture device. Abnormal fractures, concluding femoral neck fracture and distal femoral fracture, in all mice in this study were not included in the experimental results.
Procedure of the femur fracture model in mice. The constructive procedures and details of the two animal models are also described (Figure 1B, C). The type of intramedullary nails used in surgery depended on the age of the mice to ensure stable fixation of the intramedullary pins (Figure 2A, B). In this study, 6-8 weeks old mice were used, so the corresponding 26G needles were used as intramedullary pins.
Assessment of surgical quality between two fracture models. The repeatability and stability of the operation can be reflected by several objective indexes in the establishment of the bone fracture models. The operation time in the open fracture model was significantly longer than that of closed fractures (16.630±1.598 vs. 7.125±1.126, min/mouse), mainly because closed fractures do not require exposure to the femur and surgical incision suture. Although the intraoperative wound in the open fracture model was small, closed fractures had a smaller wound (8.575±0.544 vs. 0.925±0.117, mm/mouse). Regarding the modeling of the success rate, open fractures were slightly more successful than closed (96.67% vs. 95.00%). However, simultaneously, the risk of wound infection (1.67% vs. 0%) and the acute event of stride dissymmetry (3.33% vs. 0%) were slightly higher. The objective critique of post-fracture radiographs showed that the fracture lines in the open model were all in the femoral shaft; the closed model had 57 mice (totaling 60 mice) in the femoral shaft (Table I). In summary, the closed fracture model relying on the novel device had the advantages of high efficiency and repeatability compared with the traditional open fracture. Additionally, the surgical quality assessment revealed that the non-invasive procedure of the closed fracture construction process reduced injury events caused by wound infection and muscle dissection. To sum up, in terms of surgical quality, the closed fracture model had a comprehensive advantage over the open model.
Radiographic features of fracture healing (x-ray). The typical fracture healing process, biological events, and cellular activities at different stages are summarized and illustrated in Figure 3A. Fracture healing was assessed by serial radiographs at 0, 7, 14, 21, and 28 days post-fracture using six representative mice per group. Moreover, the timeline of fracture healing in the two groups underscored the reproducibility of healing in this model (Figure 3B). Radiographs at different weeks were evaluated blindly by three independent investigators using a radiographic-scoring scale system based on the bone bridge of the cortices and acceleration of healing, presented in Table II. Modified radiographic union scale in tibia (modified RUST) scoring was also used for imaging evaluation based on the callus condition in Table III. Radiographs showed that open models had no significantly larger callus area at any point post-fracture compared to the closed ones (Figure 3C). Both imaging scoring systems showed no difference in imaging features at any point post-fracture in mice of both fracture models (Figure 3D, E).
Radiographic features of fracture healing (X-Ray). (A) Illustration of a typical femur fracture healing process and biological events at different postoperation stages. The inflammatory stage (red bar) of fracture healing occurs first. The endochondral ossification stage (green bar) overlaps with the coupled remodeling stage (blue bar). The time scale of healing is equivalent to two femur fracture models fixed with an intramedullary pin. (B) The timeline of fracture healing in two groups to underscore the reproducibility of healing in this model. Dorsal-ventral radiographs of the healing femurs at 0, 7, 14, 21, and 28 days post-fracture. (C) Quantification of the callus area in fractured femurs [mean±standard deviation (SD), two-way ANOVA with Bonferroni correction, n=6 per group]. (D) Radiographic scores at 7, 14, 21, and 28 days post-fracture assessed blindly by three independent investigators using the scoring system based on the remodeling of the cortices and acceleration of healing (presented in Table II). (E) The modified RUST (RUSF) system score at 7, 14, 21, and 28 days post-fracture evaluated by the condition of the callus (presented in Table III).
Radiographic features of fracture healing (micro-CT). Micro-CT analysis revealed that vertical and transverse diameters of the callus bone volume (callus-BV) in both groups had no difference at any point post-fracture (Figure 4A). Additionally, the bone volume/tissue volume (BVcallus/TVcallus) of fractured femurs also had no difference at 14, 21, and 28 days post-fracture (Figure 4B), while there was a slight difference between the two groups at seven days, possibly due to the extra callus formation stimulated by the repair of soft tissue damage from open fractures (Figure 4C). The above results suggest that the callus area of both models peaked at 14 days and began to fall based on the maximum callus diameter, which is consistent with previous research (14). Furthermore, remodeling was prominent 21 days after fracture in both models as callus size decreased and continued through 28 days post-fracture when the external callus was gradually resorbed.
Radiographic features of fracture healing (micro-CT). (A) Representative micro-CT images of the fractured region of femurs at 7, 14, 21, and 28 days post-fracture: 3D rendering image and axial view for various treatment regimens. Axial view. (B) Quantitative measurements of callus volume and (C) bone volume/total volume in callus (BVcallus/TVcallus) at 7, 14, 21, and 28 days post-fracture in the two fracture groups [*p<0.05, mean±standard deviation (SD), two-way ANOVA with Bonferroni correction, n=6 per group]. Scale bar represents 2 mm.
The typical endochondral ossification pathway after fracture in pathological assessment. Histomorphometric analysis of HE and SO&FG stained sections from the fractured femurs of both models revealed that all fracture healing occurred via the endochondral ossification pathway, consistent with the repair mechanism after long bone fracture. Both fracture groups had no differences in the morphological changes during repair (Figure 5A). Histomorphometric analysis of SO&FG sections from the fractured femurs revealed that all fractures healed via endochondral ossification (Figure 5B). The whole callus area and cartilage area in both fracture groups decreased significantly 14 days post-fracture. The woven bone area in both fracture groups increased from 14 days post-fracture to 21 days (Figure 5C, D). qRT-PCR results of callus tissue at different healing time points showed that the expression of Col2a1 (the chondrogenic gene) in two fracture models was highest at 7 days after surgery and gradually decreased with the healing process (p=0.0047 for closed fracture models and p=0.0003 for open fracture models, Figure 5E). Interestingly, the expression of the osteogenic gene Col1a1 in two fracture models was all highest at 21 days after surgery and gradually decreased with the healing process (p=0.0305 and p=0.0086, Figure 5F). Meanwhile, the expression of osteogenesis-related proteins (osteopontin and osteocalcin) all increased gradually with the healing time during the process of bone healing in the two fracture models (Figure 5G-H). These results above indicate that cartilage morphology was most evident at 14 days after fracture, and chondrogenic repair begins at 7 days. In contrast, ossification increases throughout the healing process in the two fracture models. The fracture healing process and repair mode were consistent in both fracture models, so the reliability and accuracy of both models were guaranteed.
The typical endochondral ossification pathway after fracture in pathological assessment. (A) Hematoxylin and Eosin (H&E) staining of bone and cartilage fraction in callus at 7, 14, 21, and 28 days in two fracture groups. (B) Representative images of fractured femurs stained with 0.1% Safranin O/0.5% Fast Green (SO&FG) staining. (C) Relative cartilage area, and (D) relative woven bone area in the periosteal fracture callus at different days post-fracture [mean±standard deviation (SD), two-way ANOVA with Bonferroni correction, n=6 per group]. (E) qRT-PCR for Col2a1 and Col1a1. (F) Results of callus tissue at different healing time points in two fracture groups. (G-H) Western blot for osteopontin and osteocalcin results of callus tissue at different healing time points in the two fracture groups. Scale bar represents 2 mm. B, Bone; BM, bone marrow; WB, woven bone; M, muscle; C, cartilage chondrocytes.
The closed fracture model had a more rapid pain relief than the open model. In this study, the mice of the sham group in the closed fracture model were used with intramedullary pins without pinching off by the novel closed fracture instrument. For the open fracture model, only the skin and muscle of the mice of sham group were cut, but the femur was not severed. Paw area, stance stride, and stride length can indicate motion data as a basis for nerve damage, body load capacity, and pain level (15-17). The sciatic function index is widely used to evaluate functional recovery after sciatic nerve injury. This quantitative, non-invasive method can be used to track regenerative capacity, which is evident in the animals’ gait (18).
The experimental test showed a significant difference between the sham group and the fracture models but no difference between the open and closed fracture models in many gait indexes (Figure 6A). The results from both fracture groups showed the paw area (p<0.0001 for both, Figure 6B-C) and the percentage of stance stride (p=0.0204 for closed fracture models, and p=0.0029 for open fracture models, Figure 6E) decreased, while the stride length (p=0.0072 for closed fracture models and p=0.0014 for open fracture models, Figure 6D) and sciatic function index (SFI, p=0.0406 for closed fracture models, and p=0.0219 for open fracture models, Figure 6F) increased compared with the sham group. More interestingly, the paw area and the percentage of stance stride in the closed fracture mice were larger than those in the open group (p=0.0195 for both). The stride length was shorter than those in the open group (p=0.0072 for both). The above results showed that both fracture groups differed in gait behavior three weeks after surgery compared with the sham group. The paw area and the percentage of stance stride of mice in the closed group were increased (p=0.0195 for closed fracture models vs. open fracture models, and p=0.0204 for closed fracture models), while the stride length was closer to the sham group than the open group (p=0.0186). In conclusion, the postoperative pain symptoms of mice in the closed fracture model were milder and experienced more rapid pain relief than those in the open model, which provided modeling stability and data reliability.
The closed fracture model had a more rapid pain relief than the open model. (A) Gait mechanics and posture of the DigiGait imaging system. (B) Representative dynamic gait signals of two fracture models and the sham group; RH, right hind limb. (C) The paw area signals of two fracture models and the sham group after seven days of surgery [mean±standard deviation (SD), two-way ANOVA with Bonferroni correction, ***p<0.001, n=6 per group]. Stride length (D), % stance stride (E), and sciatic functional index (SFI, F) of the two fracture models and the sham group after seven days of surgery (mean±SD, two-way ANOVA with Bonferroni correction, *p<0.05 and **p<0.01, n=6 per group).
Discussion
Murine models have been extensively used to illustrate the biology of fracture healing and bone development. Mice are the most commonly used experimental animals in physiology and pathology (19). As the background strain of isotypic mice with spontaneous and induced mutations, mice have the advantages of high genetic background stability and consistency of experimental data generation (20, 21). However, due to the small specimen size and challenging orthopedic surgical procedures, most fracture healing studies using mice have been performed on the diaphysis of the femur via invasive operations (22) or non-invasive methods with large-sized equipment (23, 24). Although devices provide repeatable force, strike depth, and location of closed fractures (25-27), constructing closed fracture models in mice mainly relies on large weight-striker devices, which are expensive and inconvenient to move, resulting in increased modelling costs. Therefore, a convenient and efficient device for constructing closed fractures should be developed.
The ideal experimental fracture should be standardized with regard to type, site, degree of displacement, rigidity of fixation, and soft-tissue injury. This study described and elaborated on the model construction method of conventional open fractures in detail. During the construction of the open fracture model, we used dental probes as an auxiliary instrument, which significantly reduced the risk of muscle and nerve injury during surgery. We also introduced a method for constructing closed fractures with a self-designed device and compared the establishment and healing processes of the two fracture models. The new device proved highly convenient and efficient for constructing closed femur fracture models in mice. Our findings indicate that a smaller fraction of mice in the open model had symptoms of stride dissymmetry in the early postoperative period compared to the closed model, which may have been caused by the open surgical approach.
In addition, we stained calluses at different stages after fracture to determine the biological behavior of the fracture healing process. In this paper, histomorphometric analysis of safranin O-stained sections from the fractured femurs revealed that endochondral callus formation began seven days after fracture. The coupled remodeling phase started 14 days after fracture, consistent with previous studies (28). In addition, the expression analysis of extracellular matrix proteins and mRNAs corroborated these results. The expression of the chondrogenic gene Col2a1 was at its highest 7 days after surgery and gradually decreased with the healing process. The expression of the osteogenic gene Col1a1 was at its highest 21 days after surgery and gradually decreased with the healing process. Maximal osteogenic differentiation was observed between days 14 and 21, as characterized by peak levels of osteogenesis-related factor expression (osteopontin and osteocalcin), consistent with previous research (25). The results above showed the typical intrachondral osteogenic pathway of fractures in multiple dimensions in these two fracture models.
Pain is a common symptom after bone fracture fixation, and different surgical procedures cause varying pain levels. For sustained pain management in mice, the availability of a buprenorphine depot formulation for rodents would be a helpful addition, improving animal welfare (29). Evaluating pain can be a crucial indicator for judging the effectiveness of different fracture surgery models. Since animals cannot communicate their pain verbally, assessing the pain level depends more on examining behavioral changes; therefore, accurate indicators are crucial for evaluating experimental pain (30, 31). In this study, we used a small animal gait analyzer to evaluate pain symptoms after surgery by calculating the gait and paw prints of mice. The results of multiple data sets showed that postoperative pain symptoms in mice in the open fracture model were more severe than those in the closed fracture model. In other words, the closed fracture model provided more rapid pain relief after surgery than the open model. The number of mice used in each model in this research was relatively small but there was sufficient statistical power to compare between the models. Additionally, the instrument still has the potential for optimization and commercialization to suit mice of different ages.
Conclusion
Altogether, compared to traditional large modelling instruments, our novel femur fracture device provided a cost-effective and convenient strategy for establishment of the closed fracture model, and unraveled molecular mechanisms of fracture healing and tissue differentiation. Besides, the detailed explanation of surgical procedures for the open fracture model and the closed model will help researchers reconstruct fracture models in mice. Therefore, the choice of the mouse fracture model needs to take into account the advantages and disadvantages of both, scientific judgment and experimental verification. Regardless of the model chosen, reliable and convenient surgical instruments are essential for orthopedic research. The successful verification of our innovative closed fracture model device and our stable open fracture model procedure demonstrate that our two fracture models in mice have a high construction success rate and stable reproducibility.
Acknowledgements
We appreciate Dr. Yuting Yue’s contribution to the Western blotting experiments, and Zuoxing Wu’s contribution to micro-CT data extraction work for this article. The authors thank all of the members of the Ren Xu laboratory for the stimulating discussions. We thank Medjaden Inc. for scientifically editing this manuscript. This study is supported by the Natural Science Foundation of Fujian Province (2020J05008 to YC and 2020J01122649 to GR) and the National Natural Science Foundation of China (81972034 and 92068104 to RX).
Footnotes
Authors’ Contributions
LZ: drafted the paper. LZ, XS and XC: conducted experiments. GR, NL, and RX: performed substantial contributions to research design. YC: interpreted the data. All Authors: revised the paper critically and approved the submitted version.
Conflicts of Interest
All Authors declare that they have no conflicts of interest.
- Received August 9, 2023.
- Revision received October 7, 2023.
- Accepted October 16, 2023.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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