Bone fractures are a common injury with increasing incidence. While most heal quickly, a significant percentage result in delayed healing or non-unions. Current treatments, including intramedullary nails, plates, bone grafts, and BMP therapy, aren't always successful. Research focuses on understanding the cellular and molecular mechanisms of bone healing to improve regeneration. Rodent models, like mouse femur fractures, are frequently used. Mammalian bone healing involves inflammation, mesenchymal progenitor attraction, and either intramembranous or endochondral ossification depending on fracture stability. Endochondral ossification forms a cartilaginous callus, later replaced by bone. Critical-sized defects (CSDs) represent a model for non-union fractures. Axolotls, known for limb regeneration, offer a potential model for studying bone healing. While they regenerate amputated limbs, including the skeleton, they don't heal CSDs. Previous axolotl fracture studies lacked standardization, using varying animal sizes and indirect fixation methods, making comparisons difficult. This study aims to establish a reliable, standardized fracture model in axolotls using internal plate fixation, allowing comparison with mammalian models and investigation of axolotl bone healing.

Existing literature on axolotl bone healing is limited and lacks standardization. Studies using zeugopod bones (ulna/radius or tibia/fibula) with one bone serving as a fixator resulted in inconsistencies due to misaligned fragments and varying animal sizes and ages. The size of critical-sized defects (CSDs) used in previous studies varied considerably, and the indirect fixation often led to fusion with the supporting bone, complicating interpretation. These inconsistencies highlighted the need for a standardized fracture model in axolotls to facilitate comparison with mammalian models and to gain a better understanding of bone healing mechanisms in this remarkable regenerative species. Studies in mammals, especially mice and rats, employ various fixation methods, including intramedullary pins and external/internal plate fixators. Internal plates are preferred for axolotls due to their aquatic nature.

The study used 5–8-year-old axolotls (≥20 cm snout-to-tailtip length) whose femurs resembled murine femurs in size and shape. A 7.75 mm MouseFix Plate (RISystem) was used for internal plate fixation. A 0.7 mm osteotomy was created in the femur. This was compared to a non-stabilized osteotomy and limb amputation. Samples were harvested at 3 weeks, 3, 6, and 9 months post-surgery. Micro-CT, histology (Movat's Pentachrome and Safranin O/Light green), and immunohistochemistry (PCNA and SOX9) were used to assess bone healing. Mouse femur fractures (0.7 mm gap, 10 mm MouseFix Plate) were included for comparison. Axolotl bone structure was compared between young (13 cm) and old (≥20 cm) individuals and with mice. EdU labeling was used to assess cell proliferation in axolotls. Statistical analysis methods were not specified in the provided text.

Axolotl bone structure showed gradual ossification of diaphyses, with even older animals lacking secondary ossification centers. The axolotl epiphysis resembled the mammalian growth plate. Aged axolotl bone marrow contained numerous fatty vacuoles. Plate fixation successfully stabilized axolotl femur osteotomies. Plate-fixated femurs showed smaller callus formation and better fracture healing compared to non-fixated fractures. In plate-fixated fractures, bone bridging was nearly complete by 3 months, while non-fixated fractures showed misalignment and larger callus formation. Amputated limbs efficiently restored bone length and structure. Both plate-fixated and non-fixated fractures showed PCNA+ proliferating cells at 3 weeks post-injury, similar to mice. SOX9-expressing cells were observed in early stages of fracture healing and limb regeneration in axolotls, preceding cartilage formation, indicating endochondral ossification. By 9 months, non-critical osteotomies had healed, although with incomplete length restoration in the non-stabilized group. Amputated limbs were fully regenerated by 9 months. The study demonstrated that limb regeneration was faster than fracture healing even for a small, non-critical fracture.

This study established a standardized axolotl bone fracture model comparable to mammalian models. The results showed that axolotl bone healing, while using endochondral ossification like mammals, proceeds significantly slower than in mice, possibly due to slower cell proliferation rates and the unique morphology of the axolotl bone marrow. The faster regeneration of amputated limbs compared to fracture healing, even for a non-critical fracture, raises questions about the distinct regulatory mechanisms at play. The standardized model allows future studies investigating the molecular and cellular differences between axolotl limb regeneration and fracture healing and their comparison with mammalian systems. This could lead to insights for improving bone regeneration strategies in mammals.

This study successfully established a standardized, plate-fixated fracture model in axolotls, enabling controlled comparisons with mammalian bone healing. The slower healing in axolotls, compared to mice, despite similar cellular processes, highlights the need for further research into the underlying regulatory mechanisms. This model provides a valuable tool for future studies investigating axolotl bone regeneration, ultimately aiming to translate this knowledge to improve human bone fracture healing.

The study focused on a specific age group of axolotls and a single fracture size. The generalizability of these findings to younger or older axolotls and different fracture types requires further investigation. The study also lacked a detailed statistical analysis of the results, which could provide a more robust interpretation of the findings. The sample size in some experimental groups was relatively small. Furthermore, the biomechanical differences between aquatic axolotls and terrestrial mammals might play a role in the observed differences in healing times, warranting further investigation into the role of biomechanical stimuli on fracture healing.