Despite the high incidence of metaphyseal bone fractures in patients, the cellular and molecular mechanisms underlying the healing process of metaphyseal fractures are still poorly understood due to the unavailability of suitable experimental animal models. The intention of the current work was to establish a novel clinically relevant large animal model for metaphyseal bone healing in the distal femur of skeletally matured sheep. In contrast to commonly used partial osteotomy models, this model employed a complete wedge-shaped osteotomy at the metaphysis. The osteotomy was stabilised internally with a customised anatomical locking titanium plate that allowed immediate postoperative full weight bearing. Bone healing was evaluated at 12 weeks post-fracture relative to the unoperated contralateral femur. No plate breakage or secondary fracture was recorded in the animals. Histological and micro-computed tomography results revealed an increased amount of mineralised and dense trabeculae with rich bone marrow. The new trabeculae healed via direct intramembranous ossification without visible callus and cartilaginous tissue formation. Functional morphological analysis of the osteocyte-lacuna revealed regularly arranged spherically shaped osteocytes-lacunae along with the canaliculi system. The biomechanical test revealed a comparable stiffness in both operated and unoperated femurs. However, the stiffness at their cortical regions was two-fold more than in their trabecular regions. Histomorphometric assessment of two major bone matrix enzymes: ALP (osteoblast marker) and TRAP (osteoclast marker) revealed a significantly higher ALP activity in the operated femur. Bone remodelling was evident by the expressed bone formation markers (COL1A1, ALP, BGLAP, BMP2, ASMA, OPG) and bone resorption markers (RANKL, CTSK) as revealed by quantitative PCR and immunohistochemistry. Bone surface biochemical analysis using time-of-flight secondary ion mass spectrometry showed high, and a homogeneously distributed calcium and collagenous components. Ultrastructural imaging of the new trabeculae revealed a characteristic parallel arrangement of the collagen fibrils uniformly mineralised by the dense mineral substance. Active osteoblasts were characterised by their plump cuboidal-shape, abundant rough endoplasmic reticulum, and a distinct Golgi apparatus whereas active osteoclasts were characterised by their large size, multiple nuclei and a ruffled border. The ultrastructure of the osteocytes varied according to the mineralisation level. The matured star-shaped osteocytes embedded inside the fully mineralised bone whereas young osteocytes embedded within the osteoid or partially mineralised bone areas. In conclusion, this newly established metaphyseal fracture model is of interest to study bone healing and treatment options for the enhancement of metaphyseal fractures.
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