BMDMs in metabolic memory impair fracture healing in diabetes

Abstract

Background: The risk of fractures nonunion and delayed union in diabetes mellitus remains elevated despite glucose-lowering therapies. We hypothesized that bone marrow-derived macrophages (BMDMs) can be induced in the status of metabolic memory and still impair fracture healing when hyperglycemia stimulus disappears. Methods: Diabetic mice were divided into control (Ctrl), diabetic (DM), and diabetic with glucose control (DM/GC) groups. Fracture healing was assessed by micro-CT and histology, evaluating callus volume, bone volume/total volume (BV/TV), and inflammatory markers. In vitro, bone marrow-derived macrophages (BMDMs) were exposed to high glucose (HG) for varying periods to simulate hyperglycemia-induced metabolic memory, followed by normalization. Pro-inflammatory cytokines and macrophage polarization (M1/M2) were assessed via ELISA and flow cytometry. Osteogenesis and angiogenesis were evaluated in co-culture assays. RNA-seq and ATAC-seq were performed to analyze gene expression and chromatin accessibility, focusing on inflammatory pathways and CEBPB. Results: All data show that BMDMs play a significant role in the sustained effects of hyperglycemia on fracture healing even after glucose normalization in diabetic animals. Hyperglycemia-induced metabolic memory in BMDMs resulted in increased pro-inflammatory cytokines and a higher proportion of M1 macrophages, which impaired osteogenesis and angiogenesis. The co-culture medium from BMDMs in metabolic memory conditions suppressed osteogenesis in BMSCs and angiogenesis in HUVECs. Integrated analysis of RNA-seq and ATAC-seq in BMDMs revealed that inflammatory pathways were upregulated, with CEBPB identified as a key factor. Silencing CEBPB reversed these adverse effects and enhanced fracture healing in a diabetic model. Conclusions: Our results demonstrate the reason why the glucose-lowering therapies is unsuccessful in reducing the risk of fractures nonunion and delayed union in patients with diabetes mellitus, and shed light on a new strategy for the disease.