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PURPOSE OF THE STUDY The preclinical study aimed to compare the healing of segmental bone defects treated with biodegradable hyaluronic acid and tricalcium phosphate-based hydrogel with the established autologous spongioplasty. Another aim was to evaluate the hydrogel as a scaffold for osteoinductive growth factor of bone morphogenetic protein-2 (BMP-2) and stem cells. MATERIAL AND METHODS The study was conducted in an in vivo animal model. A standardized rabbit model of a 15 mm long segmental bone defect of left radius was used. A total of 40 animals were divided into 5 groups of 8 individuals. In the KO- (negative control) group, the created defect was left to heal spontaneously. In the KO+ (positive control) group, the defect was filled with morselized bone autograft prepared from the resected segment. In the study group A, the defect was filled with hydrogel based on hyaluronic acid derivative and tricalcium phosphate. In the study group B, the defect was filled with hydrogel based on hyaluronic acid derivative, tricalcium phosphate and bone marrow aspirate. In the study group C, the defect was filled with hydrogel based on hyaluronic acid derivative, tricalcium phosphate, bone marrow aspirate and BMP-2. Healing was assessed using radiographs at 1, 6, and 12 weeks postoperatively and histology specimens were collected at 16 weeks postoperatively. RESULTS Altogether 35 rabbits survived (KO- 7, KO+ 7, A 7, B 6, C 8) until the end of the study. As concerns the radiographic assessment, the best results were achieved by the groups KO+ and C, where new bone formation across the entire width of the bone defect was clearly seen at 6 and 12 weeks and the osteotomy line was completely healed too. At 12 weeks, complete bone remodelling was observed in all animals in the group KO+, whereas in the group C, bone remodelling was fully completed in 5 animals and partially completed in 3 animals. In terms of histological assessment, however, the best results were achieved by the group C, where the bone defect was completely remodelled into lamellar bone in 7 specimens, while in 1 specimen it healed with bony callus formation. In the group KO+, the defect was healed in 4 specimens by cartilaginous callus with loci of remodelling into bony callus, in 2 specimens the bony callus was predominant with cartilaginous callus areas, and only one defect was completely remodelled into lamellar bone. DISCUSSION Compared to autografts that manifest osteogenic, osteoinductive and osteoconductive properties, the biodegradable hyaluronic acid and tricalcium phosphate-based hydrogel has osteoconductive properties only. Thus, it was also tested in our study as a scaffold for bone marrow cells and BMP-2 osteoinductive growth factor. Thanks to its semi-liquid properties, the biodegradable hyaluronic acid and tricalcium phosphate-based hydrogel is a promising material for use in 3D printing. CONCLUSIONS The preclinical study in an in vivo animal model confirmed the beneficial effect of the biodegradable hyaluronic acid and tricalcium phosphate-based hydrogel on the healing of critical-size segmental bone defects. Better healing of these defects was also confirmed for filling composed of hydrogel and BMP-2 osteoinductive growth factor. The benefit of bone marrow aspirate mixed with hydrogel was not confirmed. [ABSTRACT FROM AUTHOR]