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A novel mouse model to study fracture healing of the proximal femur

The majority of fractures, especially in elderly and osteoporotic patients, occurs in metaphyseal bone. However, only a few experimental models exist to study metaphyseal bone healing in mice. Currently used mouse models of metaphyseal fracture healing are either based on drill hole defects, lacking adequate biomechanical stimulation at the site of fracture and therefore endochondral ossification in the fracture callus, or are introduced into the distal part of the mouse femur stabilized by a locking plate, which is challenging due to the small specimen size. Therefore, the aim of the current study was to develop a new mouse model to study metaphyseal fracture healing of the proximal femur. We chose a combination between an open osteotomy and a closed intramedullary stabilization. A 24 G needle was inserted into the femur in a closed manner, then an osteotomy was made with a 0.4‐mm Gigli wire saw between the third and the lesser trochanter of the femur using an open approach. Fractured femurs were analyzed using microcomputed tomography and histology at days 14 and 21 after surgery. No animals were lost due to surgery or anesthesia. All animals displayed normal limb loading and a physiological gait pattern within the first three days after fracture. We found robust endochondral ossification during the fracture healing process with high expression of late chondrocyte and early osteogenic markers at day 14 (d14). By day 21 (d21), all fractures had a bony bridging score of 3 or more, indicating successful healing. Callus volume significantly decreased from d14 to d21, whereas high numbers of osteoclasts appeared at the fracture callus until d21, indicating that callus remodeling had already started at d21. In conclusion, we successfully developed a novel mouse model to study endochondral fracture healing of the proximal femur. This model might be useful for future studies using transgenic animals to unravel molecular mechanisms of osteoporotic metaphyseal fracture healing.

Therefore, the aim of the current study was to develop a new mouse model to study metaphyseal fracture healing of the proximal femur. We chose a combination between an open osteotomy and a closed intramedullary stabilization. A 24 G needle was inserted into the femur in a closed manner, then an osteotomy was made with a 0.4-mm Gigli wire saw between the third and the lesser trochanter of the femur using an open approach. Fractured femurs were analyzed using microcomputed tomography and histology at days 14 and 21 after surgery. No animals were lost due to surgery or anesthesia. All animals displayed normal limb loading and a physiological gait pattern within the first three days after fracture. We found robust endochondral ossification during the fracture healing process with high expression of late chondrocyte and early osteogenic markers at day 14 (d14). By day 21 (d21), all fractures had a bony bridging score of 3 or more, indicating successful healing. Callus volume significantly decreased from d14 to d21, whereas high numbers of osteoclasts appeared at the fracture callus until d21, indicating that callus remodeling had already started at d21. In conclusion, we successfully developed a novel mouse model to study endochondral fracture healing of the proximal femur. This model might be useful for future studies using transgenic animals to unravel molecular mechanisms of osteoporotic metaphyseal fracture healing.

| INTRODUCTION
The majority of fractures, especially in elderly and osteoporotic patients, occurs in metaphyseal bone due to the susceptibility of trabecular bone to microstructural damage. 1,2 These fractures are challenging to treat because the weak bone quality limits proper osteosynthesis fixation and there is evidence for reduced healing capacity of osteoporotic bone. 3 Furthermore, recent research has demonstrated that there are significant differences between the fracture healing process in metaphyseal and diaphyseal bone. Tätting et al 4 revealed increased numbers of lymphocytes and monocytes and decreased numbers of granulocytes when comparing the inflammatory response after fracture in diaphyseal vs metaphyseal bone. Further studies demonstrate different effects of both antiresorptive and osteoanabolic treatment strategies including Wnt/β-catenin activation, parathyroid hormone application, and bisphosphonate injections on metaphyseal and diaphyseal bone healing. 5,6 Therefore, metaphyseal osteoporotic fractures require more complex strategies to facilitate optimal bone healing. This is especially true for osteoporotic hip fractures, which occur mainly in the region of the femoral neck and the femoral trochanter and are therefore classified as metaphyseal fractures.
While these injuries are important from a clinical standpoint, adequate small animal models to study them are lacking. In fact, only a few animal models exist to study metaphyseal bone healing, and the majority of these studies have been performed in sheep, dogs, and rats, limiting the possible use of genetically modified animals to unravel molecular mechanisms involved in bone regeneration. 1 For this purpose, mouse models to study metaphyseal fracture healing are desirable because of the large number of available knock-out, knockin, and transgenic mouse strains. 3 Indeed, 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 or tibia. Currently used mouse models of metaphyseal fracture healing are either based on drill hole defects, lacking adequate biomechanical stimulation at the site of fracture and therefore endochondral ossification in the fracture callus, 7 or are introduced into the distal part of the mouse femur stabilized by a locking plate, which is challenging due to the small specimen size. 8 Therefore, to begin to understand the molecular mechanisms of fracture healing and investigate possible treatment options in a more clinically relevant manner, a robust and reliable mouse model of metaphyseal fracture healing would be useful. Therefore, the aim of the current study was to develop a novel mouse model to study metaphyseal fracture healing of the proximal femur.

| Animals and experimental design
All animal experiments were approved by the local animal welfare committee (IACUC UCSF AN143402-03B) and were performed in compliance with international regulations for laboratory animal welfare and handling (ARRIVE guidelines). Twelve-week-old female C57BL/6J mice were purchased from Jackson laboratories. Young mice were chosen for this study to determine the feasibility of the new surgery technique and to investigate the physiological healing process of proximal femur fractures in mice. Mice were housed in groups of up to five animals with free access to standard mouse diet and water. The surgical procedure was conducted under general anesthesia using ketamine hydrochloride (100 mg/mL; 50 mg/kg bodyweight; Henry Schein Animal Health) at a ratio of 1:1 with dexmedetomidine hydrochloride (0.5 mg/mL; Orion Pharma) and analgesia using buprenex (0.03 mg/mL; 0.05 mg/kg bodyweight). Buprenex was injected subcutaneously every 6 hours for the first 24 hours. Mice were allowed to move freely directly after surgery.
Animals were monitored daily to investigate nonphysiological or physiological limb loading and gait pattern. After 14 and 21 days, mice were euthanized using carbon dioxide and cervical dislocation.
Fractured femurs were harvested and fixed in 4% paraformaldehyde for 48 hours. Afterward, implant material was removed and fractured femurs were subjected to microcomputed tomography (µCT) scans and decalcified histology. The sample size was 6 per time point.

| Surgical procedure
A 24 G needle was introduced retrograde into the right femur according to previously established procedures 9 ( Figure 1). For femur osteotomy, a medial 0.5-cm skin incision was made longitudinally along the femur.
The femoral muscles were separated bluntly and the tendon insertion at the third trochanter was cut to allow free access to the proximal part of F I G U R E 1 Verification of success of the fracture surgery. Representative X-ray image of a fractured femur directly after surgery the femur. The osteotomy was sawed between the third and the lesser trochanter using a 0.44-mm Gigli wire saw ( Figure 1). The bone was sawed from the bottom to the top until the saw hit the needle, then the sawing direction was changed to top-bottom until the osteotomy was complete. After flushing the osteotomy gap with sterile sodium chloride, the muscles were sutured together using a continuous U-suture with absorbable Vicryl 5-0. The skin was sutured using an interrupted stitch with nonabsorbable Resolon 5-0.

| Histological analysis
After µCT scans, right femurs were subjected to decalcified histology as described previously. 11  The region of interest was whole fracture callus.

| Statistical analysis
Data were tested for normal distribution using Shapiro-Wilk normality test. Most data sets were normally distributed. Therefore, results are presented as dot plots with mean ± standard deviation.
Statistical analysis was done by Student's t test (GraphPad Prism 9).
The level of significance was set at P < .05. The group size was six, and was calculated based on the findings of a previous fracture healing study with the main outcome parameters flexural rigidity and BV/TV in the fracture callus. 13

| RESULTS
No animals were lost due to surgery or anesthesia. All animals displayed normal limb loading and a physiological gait pattern within the first 3 days after fracture.
µCT analysis revealed successful osteotomy between the lesser and the third trochanter in all animals (Figure 2A,B). Bony bridging score increased significantly between day 14 (d14) and day 21 (d21) from values 0 or 1 at d14 to values 3 or 4 at d21 ( Figure 2C). BV ratio also increased significantly between d14 and d21 ( Figure 2D). Total BV did not differ between the different time points, however, total callus volume decreased significantly between d14 and d21 ( Figure 2E,F). Furthermore, whole callus MMIx decreased significantly, whereas BMD of the bony callus increased significantly between d14 and d21 ( Figure 2G   femur of mice is not the same as in humans and therefore the biomechanical environment might differ. Furthermore, the stabilization by a cannula is inexpensive and relatively easy to place into the intramedullary canal, and importantly, it did not appear to cause sustained pain. In conclusion, we successfully developed a novel mouse model to study endochondral fracture healing of the proximal femur.
The limitations of our model are that stabilization by a cannula did not provide rotational stability as is achieved when proximal femur antirotational nails or plates are used. Furthermore, many osteoporotic human fractures occur at the femoral neck and it is not possible to replicate this in mice due to a lack of adequate fracture implant devices for the mouse hip. Another limitation is that there are only a few trabecular bone structures in the area between the third and the lesser trochanter, therefore the real "metaphyseal" area in the mouse proximal femur is very small.
To date, there are only a few published studies of metaphyseal fracture models in mice. Most of these studies used drill hole defects in the mouse femur or tibia. [4][5][6][7]14 However, drill hole defects lack biomechanical stimulation at the fracture site and furthermore, no fracture implants are used for this approach. This might critically influence the type of healing. Furthermore, the inflammatory response is likely to be different after osteotomy or fracture due to the different amounts of tissue injury. As it was shown in diaphyseal fracture models that the inflammatory and angiogenic response might be altered during osteoporotic fracture healing, [15][16][17][18][19][20] it may be of critical importance to use a metaphyseal healing model with a certain amount of tissue injury to study this in more molecular detail. An alternative mouse model to study metaphyseal fracture healing used a plate at the distal mouse femur. 8,21 However, positioning this plate in the trabecular metaphyseal region could be challenging due to the small specimen size.
Comparing the data from our novel mouse model to published data from a diaphyseal fracture healing study using the same age, sex, and strain of mice (female, 12-week-old C57BL/6J mice), 13 it is obvious that total callus volume and BV in the fracture callus are significantly higher at d21 with our novel approach. This might be due to a different time course of healing or to different biomechanical conditions caused by different fracture implants used.
BV/TV seems to be comparable between the study from Wehrle et al and the present study. Both models showed a significant amount of cartilage in the intermediate phase of healing, indicating endochondral ossification during callus maturation. Comparing our data with other studies investigating metaphyseal fracture healing in mice, 8 it is obvious that stabilizing a distal femur fracture with a plate led to intramembranous ossification in the fracture callus in contrast to endochondral ossification seen in our model. Furthermore, callus size was significantly smaller. This might be due to the different locations of the fracture and/or due to the different biomechanical conditions caused by different fracture implants used. Also, the study by Histing et al used CD-1 mice, therefore the results are not directly comparable with our data from the present study.
In conclusion, our novel model provides a fast, reliable, and inexpensive way to study endochondral metaphyseal fracture healing in mice. Future studies using osteoporotic mice in combination with our model might help unravel molecular mechanisms of delayed osteoporotic fracture healing. We would expect differences in the healing process and healing time in aged and/or ovariectomized mice compared with nonosteoporotic animal, therefore this model might be used to determine the cellular and molecular mechanism of delayed osteoporotic fracture healing in metaphyseal bone.