Accelerated Calvarial Healing in Mice Lacking Toll-Like Receptor 4

Abstract

The bone and immune systems are closely interconnected. The immediate inflammatory response after fracture is known to trigger a healing cascade which plays an important role in bone repair. Toll-like receptor 4 (TLR4) is a member of a highly conserved receptor family and is a critical activator of the innate immune response after tissue injury. TLR4 signaling has been shown to regulate the systemic inflammatory response induced by exposed bone components during long-bone fracture. Here we tested the hypothesis that TLR4 activation affects the healing of calvarial defects. A 1.8 mm diameter calvarial defect was created in wild-type (WT) and TLR4 knockout (TLR4−/−) mice. Bone healing was tested using radiographic, histologic and gene expression analyses. Radiographic and histomorphometric analyses revealed that calvarial healing was accelerated in TLR4−/− mice. More bone was observed in TLR4−/− mice compared to WT mice at postoperative days 7 and 14, although comparable healing was achieved in both groups by day 21. Bone remodeling was detected in both groups on postoperative day 28. In TLR4−/− mice compared to WT mice, gene expression analysis revealed that higher expression levels of IL-1β, IL-6, TNF-α,TGF-β1, TGF-β3, PDGF and RANKL and lower expression level of RANK were detected at earlier time points (≤ postoperative 4 days); while higher expression levels of IL-1β and lower expression levels of VEGF, RANK, RANKL and OPG were detected at late time points (> postoperative 4 days). This study provides evidence of accelerated bone healing in TLR4−/− mice with earlier and higher expression of inflammatory cytokines and with increased osteoclastic activity. Further work is required to determine if this is due to inflammation driven by TLR4 activation.

Toll-Like Receptor 4 Mediates the Regenerative Effects of Bone Grafts for Calvarial Bone Repair

“…[30] In a previous study, accelerated bone healing within a calvarial defect model was observed in TLR4 -/-mice, suggesting that TLR4 signaling is detrimental to the normal healing of calvarial bone defects. [21] In contrast to our previous findings, we report here that TLR4 is required to support graft-induced bone repair using a calvarial defect model. At day7 after implantation, WT mice showed more F4/80 positive macrophage lineage cell infiltration, while macrophages were not detected in TLR4 -/-mice after bone component implantation.…”

“…It has been argued that the regenerative potential of bone graft is dependent upon the efficient transfer of progenitor cells to the defect site, [36,37] although donor cells typically fail to survive beyond hours to weeks. [38,39] In our previous work, [21] we used radiographic bone measurement to assess bone repair in a calvarial defect model and observed that 33.14% of the defect had regenerated by day 28 in WT mice, while the current study showed that BC and ME (matrix-enriched) groups achieved 74.80% and 66.79%, respectively (Fig.6). The CE (cellenriched) implant, in contrast, failed to stimulate bone repair beyond control values.…”

“…Fluorescence intensity was later measured using Synergy H1 Hybrid microplate reader (BioTek, Winooski, VT). 8 8 A circular parietal defect was made using a 1.8 mm outer diameter trephine (Fine Science Tools, Foster City, CA) as previously described [21] to model craniofacial bone defects that result from trauma, craniofacial reconstruction, or management of tumors. Implants were created using a 5 mm outer diameter trephine (Fine Science Tools, Foster City, CA).…”

“…[21] Calvarial samples obtained from each group were harvested, fixed in 4% paraformaldehyde, Immunohistochemical staining was performed as previously described. [21] Sections were …”

“…[17][18][19][20] In a previous study, we showed that calvarial bone healing is accelerated in TLR4 knock-out (TLR4 -/-) mice and is associated with elevated expression of cytokines and osteoclast differentiation markers. [21] Furthermore, our team has previously presented data suggesting that morselized bone graft induces a TLR4-dependent systemic inflammatory response in a bilateral femoral fracture model. [22] Based upon these observations, we hypothesized that TLR4 signaling is necessary for graft-induced bone formation by mediating the remodeling and integration of bone graft within regenerating calvarial defects.…”