Impaired Bone Regeneration in a Rat Model of Posttraumatic Lymphedema

Abstract

Posttraumatic lymphedema is a frequent yet underrecognized complication of severe limb trauma and fractures. Although chronic inflammation is known to impair bone repair, it is entirely unknown whether lymphatic dysfunction itself affects skeletal regeneration. To date, no clinical or experimental data exist that directly link lymphedema to impaired bone healing. Using a validated rat model including both male and female animals, tibial bone defects were created with or without concomitant lymphatic vessel disruption. Bone regeneration was assessed by histology (aniline blue), Osteocalcin immunohistochemistry, and high-resolution micro-computed tomography (μCT). Mechanistic analyses included markers of osteoblast differentiation (RUNX2), remodeling-associated signaling (RANKL), endothelial/angiogenesis-associated signaling (PECAM-1), and cellular proliferation (PCNA), and multiplex Luminex-based cytokine and chemokine profiling.

Posttraumatic lymphedema significantly impaired bone regeneration, evidenced by reduced osteoid formation, diminished Osteocalcin expression, and lower bone volume fraction on μCT (p = 0.0246). The PTL phenotype was associated with reduced osteoblast differentiation (RUNX2↓, p = 0.0038), reduced RANKL expression, consistent with altered remodeling-associated signaling (p = 0.0002), and decreased PECAM-1 expression (p = 0.0026), while PCNA expression remained unchanged. Luminex analysis revealed a distinct proinflammatory and chemotactic signature characterized by elevated TNF-α, IL-6, IL-12p70, MIP-1α, RANTES, MCP-1, and MIP-2.. This cytokine and chemokine upregulation occurred in both lymphedema groups – regardless of whether a bone defect was present – whereas animals with isolated bone defects showed only minimal inflammatory activation.

This study provides experimental evidence that posttraumatic lymphedema is associated with impaired bone regeneration. The PTL phenotype was accompanied by a sustained proinflammatory and chemotactic microenvironment, reduced osteoblast differentiation, altered remodeling-associated signaling, and reduced PECAM-1 expression. These findings identify a previously unrecognized association between posttraumatic lymphatic dysfunction and skeletal repair and provide a basis for further mechanistic investigation.