EFFECTS OF RECOMBINANT HUMAN BMP-2 AND IMMUNOMODULATION TARGETING IL-1β ON FRACTURE HEALING IN A FEMUR SEGMENTAL DEFECT MODEL IN RATS

Immunomodulation may improve the efficiency of lower and saver doses of bone morphogenetic growth factors like BMP-2, which induce inflammation/heterotopic ossification in muscle at clinically-applied doses. This study investigates (1) low (1/5µg) and high (45µg) BMP-2 on callus formation/IL-1β-production in fracture repair tissue/adjacent muscle at early time in a rat femur segmental defect model and (2) the administration of a monoclonal anti-IL-1ß antibody combined with 1μg BMP-2 on bone healing over 14 weeks. 2mm internally-fixed femoral defects were created in female Fischer rats (22–27 weeks-old) (animal license: GR/19/2022) using established protocols for analgesia+anesthesia. To study BMP-2-mediated effects on early healing, animals (n=3/group) received an empty defect, a Lyostypt collagen sponge, or Lyostypt+1/5/45µg BMP-2 (InductOs, Medtronic) and were sacrificed at days 3, 7 or 14. Radiographs were taken throughout the study. The fracture repair tissue+adjacent muscle were collected in T-PER (Thermo Fisher) and IL-1β levels determined by ELISA (R&D-Systems DuoSet). In a follow up study, animals (n=7/group) received either Lyostypt or Lyostypt+1μg BMP-2±anti-IL-1ß antibody (BioXCell, 10 mg/ml), starting from post-OP day3 and then every 3 rd day until day15. In vivo micro-CT was performed throughout the study. The mechanical properties of operated femurs were assessed by 4-point-bending (Instron5866) and compared to contralateral femurs (one-way ANOVA, GraphPad Prism8). During early healing, radiographs demonstrated a dose-dependent effect on bone formation 2 weeks post-OP for the BMP-2 groups. Lyostypt+45µg BMP-2 induced a 1.5-fold increase in IL-1β on day3 (322±32pg/ml) compared to all other groups (227±34pg/mg; day3) (Fig.2A), all groups exhibited significantly lower IL-1β levels in the adjacent muscle (p<0.001). In the second study, operated femurs with 1μg BMP-2 reached an average stiffness of 91±37% of contralateral femurs, femurs in the anti-IL-1ß group 111±12%. Micro-CT showed that Lyostypt led to 33±16% of the defect being filled with new bone after 14 weeks, 1μg BMP-2 to 66±11%, 1μg BMP-2+anti-IL-1β to 75±4%. Low and high doses of human recombinant BMP-2 had strong local effects on new bone formation, only the clinically applied dose induced an inflammation pulse at the fracture site. Combined immunomodulatory treatment with anti-IL-1ß improved functional healing outcome, (+ 20% in bone stiffness), although not significantly compared to 1µg BMP-2 alone. Future work explores the efficacy of anti-IL-1β in a diabetic bone healing environment with BMP-2. Acknowledgments: AO foundation. Technical support: Andrea Furter, Alisa Hangartner, Thomas Krüger. For any figures or tables, please contact the authors directly.