(A) Untargeted metabolomic profiling was performed on serum collected from rats subjected to either bone defect (BD) or induced membrane technique (IMT) procedures. Metabolites were considered significant if they met all 3 criteria: P < 0.05, FDR < 0.2, and Fold > 1.5. (B) In the BD model, 2-dimensional micro-CT scans were performed weekly to monitor bone stability at the 3.0 mm femoral defect site (n = 7). (C) Serum metabolite patterns were visualized through partial least squares discriminant analysis (PLS-DA) using samples from week 0 (BD:W0, n = 7, 3 rats were excluded due to hemolysis), week 1 (BD:W1, n = 7), and week 6 (BD:W6, n = 7). Each point represents an individual rat. (D) Metabolic pathway enrichment for BD rat serum was assessed using MetaboAnalyst (Hypergeometric Test). Pathways were prioritized based on significance level, represented as –log₁₀(P value), with those to the right of the red threshold line considered statistically relevant. (E) For IMT-treated rats, weekly 2D micro-CT images were acquired to track femoral changes following implantation and membrane induction (n = 6). (F) PLS-DA was also applied to serum samples from IMT rats at 3 time points: week 0 (IM:W0, shared with C), week 1 (IM:W1, n = 3), and week 6 (IM:W6, n = 3), with each dot representing one rat. (G) Metabolomics pathway enrichment for IMT serum samples was performed using MetaboAnalyst (hypergeometric test), highlighting altered pathways over time. Only pathways to the right of the red dashed significance threshold line were retained. Week 0 represented in this figure should be 10 rats, from which 3 were excluded due to hemolysis causing incorrect results of metabolomics analysis. Arg, arginine; Pro, proline; Phe, phenylalanine; Tyr, tyrosine; Trp, tryptophan.