Mechanisms responsible for the ability of enoxaparin sodium to inhibit inflammatory responses in the immune microenvironment of bone repair: A transcriptomic sequencing study

Enoxaparin sodium (ES), a low molecular weight heparin derivative, has recently been recognized for its diverse biological activities. In particular, the ability of heparin to modulate inflammation has been utilized to enhance the biocompatibility of bone implant materials. In this study, we utilized poly (methyl methacrylate) (PMMA), a drug loading bone implant material, as a matrix and combined this with enoxaparin sodium (ES) to create enoxaparin sodium PMMA cement (ES-PMMA) to investigate the regulatory effects of ES on inflammatory responses in bone tissue from an animal model. We established a rabbit model of femoral condyle bone defects to investigate the immunoregulatory mechanisms of ES-PMMA. Rabbits were divided into control (n = 5), model (n = 10), PMMA (n = 10) and ES-PMMA (n = 10) groups. The control group underwent sham surgery as a blank control, while the model group was established with a bone defect model in the rabbit femoral condyle. The PMMA group and ES-PMMA group followed the same modeling procedure as the model group. After successful modeling, the PMMA group and ES-PMMA group were implanted with PMMA bone cement columns and ES-PMMA bone cement columns, respectively. Ten days post-surgery, cancellous bone tissue from the defect site was collected from each group, and the control group was sampled at the same location. Tissue samples were collected from each group for transcriptomic sequencing. RNA sequencing (RNA-seq) was performed and differentially expressed mRNAs were identified between the model and controls, between the PMMA and model groups, and between the ES-PMMA and model groups. Key candidate genes associated with ES-PMMA treatment were identified (304 genes), and Gene Set Variation Analysis (GSVA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the differentially expressed genes and key candidate genes in each group (P < 0.05). The 304 key candidate genes associated with ES-PMMA treatment are involved in functions such as inflammation, cell proliferation, and differentiation. Protein-protein interaction (PPI) network analysis and machine learning revealed three key candidate genes in the ES-PMMA group: recombination activating gene ( RAG1 ), Src-like adaptor 2 ( SLA2 ), S100 calcium binding protein and beta (neural) ( S100B ). SLA2 and RAG1 are known to be related to inflammation, whereas S100B is related to osteogenic differentiation. Finally, the subcellular localization and functional similarities of the three genes were assessed, and their transcription factors and miRNAs were predicted. Collectively, these findings provide insights into the mechanism of ES in regulating immune responses in the bone; this may facilitate the development of novel bone implant materials.