Background and Hypothesis: High-energy fractures associated with severe soft tissue injury have a significant incidence of delayed or non-union. The soft tissue envelope may adversely contribute to the healing of a fracture, not only in stripping of the periosteal blood supply, development of compartment syndrome or tissue interposition between the bone ends but also in its ability to generate an intense acute inflammatory response. Inflammation is the initiator of healing; in clinical scenarios of impaired inflammation (immune deficiency, NSAIDs, corticosteroids) healing is delayed; interestingly, in injury with excess inflammation (CVA, MI) healing is also delayed. Would the inflammatory response following high-energy fractures contribute beneficially or adversely to the healing of the underlying fracture? Using an in-house murine femoral fracture model which reliably demonstrated features of delayed fracture healing when associated with a severe overlying muscle crush injury we proposed these hypotheses:

That fracture callus with overlying muscle crush would contain raised expression of acute inflammatory cytokines (IL-1β, IL-6 and TNF-α).

Methods: Total RNA was extracted from normal fracture callus (FO) and muscle crush fracture callus (MC) at day 2, day 4 and day 8. Semi-quantitative RT-PCR was used to compare IL-1β, IL-6 and TNF-α mRNA expression. Histomorpometric analysis of ICC stained sections of the FO and the MC groups was used to estimate IL-1β, IL-6 and TNF-α protein expression within the callus. Positively staining areas for the cytokine within the callus were a semi-quantified and compared between groups. Finally, blocking antibodies to IL-1β and TNF-α were injected into MC fracture callus at day 0, 4 and 8. Control MC group had vehicle only injected. Fracture healing was measured using radiological, histomorphological and biomechanical outcome measures. Following a pilot dosing experiment, the effect of blocking antibodies on fracture healing was compared between MC and MC with antibody groups.

Results: The MC group IL-1β mRNA expression was significantly higher than FO at day 4 and day 8 (p=0.05). ICC for IL-1β protein expression was higher on day 4 and on day 8 in the MC group, significant at day 8 (p=0.03). TNF-α mRNA expression in the MC group at day 8 was significantly higher than the FO group (p=0.05). ICC for TNF-α protein in the MC group peaked at day 8 and was significantly higher than the FO group (p< 0.03). IL-6 mRNA expression was significantly raised in the MC group at day 4 and 8 compared with the FO group (p=0.05). ICC for IL-6 protein showed significantly increased expression at day 8 in the MC group (p=0.05). The patterns of expression of the mRNA and proteins were similar.

Injection of anti-TNF-α antibodies into MC mice caused more new bone formation on day 16 (p=0.03) and day 24 (p=0.06), stiffer calluses at day 24 (p=0.01) and faster fracture gap obliteration at day 16 (p=0.05) and day 24 (p=0.001). IL-1β blockade had slightly less effect, more new bone formationd ay 16 (p=0.01) and day 24 (p=0.03), slightly stiffer (p=0.08), but no significant difference in fracture gap obliteration from controls.

Conclusion: The effect of muscle crush around the fracture callus was to increase and prolong the expression of inflammatory cytokines with the callus. The effect of blocking these excessive inflammatory cytokines in our model was to improve fracture healing. Excessive inflammatory cytokines (IL-1β, IL-6, TNF-α) in bone impair new bone production by osteoblasts, inhibit the recruitment and differentiation of mesenchymal precursors and promote osteoclastogenesis. The mechanism of action of blocking antibodies may be due to inhibition of the antiosteogenic effects of these cytokines.