Fig. 3. Plate stabilization retains bone alignment and leads to smaller callus formation with quicker transition to woven bone.

Amputated upper hind limb (left), plate-fixated (middle), and non-fixated (right) femur fracture 3 weeks (a–c), 3 (d–f), 6 (g–i) and 9 (j–l) months after bone injury. Micro-CT analysis (left panels) and tissue sections stained after Movat’s pentachrome protocol (right panels) are shown (mineralized tissue=yellow; cartilage=green, muscle=orange). a 3 weeks post-amputation, a regeneration blastema is formed at the tip of the leg stump. b In plate-fixated sample, fracture gap is clearly visible at micro-CT image. c. The non-fixated samples show severe bone misalignment. d Blastema at 3 months post-amputation. Dashed line – amputation plane in d, g, j. e Stable fixation with the bone-aligning plate provides stability and allows for formation of a cartilage sleeve bridging the bone gap. Note incomplete cortex beyond the fracture gap on the right side (red asterisk). f In non-fixated fracture, a cartilage front formed but the bone fragments are not aligned in the proper axis. The medullary cavity sealed on both sides of the fracture gap, indicating terminated bone formation at the fracture ends (f’, yellow arrowhead). g 6 months post-amputation, skeletal elements re-grow. The amputation plane—dashed line. h Plate-fixated bone improves the cortical thickness and stability by growing a second cortex (black asterisk in h’, k’). i Delayed mineralization and longer persistence of cartilage in the callus of the non-fixated fracture. j 9 months post-amputation, limb is fully regenerated (j’, blue arrowhead—secondary cortex). k Plate-fixated samples 9 months post-fracture with developed ossified hard callus, secondary cortices and fat vacuoles-filled bone cavity. l The non-stabilized fracture achieved bony bridging of the gap—the osteotomy ends of the bone showed a rounded appearance (l’, green arrowhead). Scale bars 1 mm.