Dynamics of Early Signalling Events during Fracture Healing and Potential Serum Biomarkers of Fracture Non-Union in Humans

Abstract

To characterise the dynamic of events during the early phases of fracture repair in humans, we investigated molecular events using gene expression profiling of bone fragments from the fracture site at different time points after trauma and immune/stromal cells recruitment at the fracture site using flow cytometry. Bone and inflammatory markers were expressed at low levels at homeostasis, while transcripts for bone constituent proteins were consistently detected at higher levels. Early after fracture (range 2–4 days), increased expression of CXCL12, suggested recruitment of immune cells associated with a change in the balance of degradation enzymes and their inhibitors. At intermediate time after fracture (4–8 days), we observed high expression of inflammatory cytokines (IL1-beta, IL6), CCL2, the T-cell activation marker CD69. Late after fracture (8–14 days), high expression of factors co-operating towards the regulation of bone turnover was detected. We identified potential soluble factors and explored circulating levels in patients for whom a union/non-union (U/NU) outcome was known. This showed a clear difference for PlGF (p = 0.003) at day 1. These findings can inform future studies further investigating the cascade of molecular events following fractures and for the prediction of fracture non-union.

Influence of the TGF-β Superfamily on Osteoclasts/Osteoblasts Balance in Physiological and Pathological Bone Conditions

“…Endochondral process occurs in three major phases—inflammation and hematoma formation, then bone repair (fibrocartilaginous and bony callus formation), and finally bone remodeling [ 104 ]. During the phase of inflammation and hematoma formation, the platelet–fibrin clot acts as a transitory scaffold that is able to recruit cells involved in the acute inflammation, via the presence of cytokines, such as IL-1 and IL-6, as well as chemoattractants like CXCL12 [ 105 , 106 , 107 ]. Interestingly, Burska et al recently found an increase in IL-1β and IL-6, but not in TNF-α, during the early hematoma and inflammation phase in humans.…”

“…Interestingly, Burska et al recently found an increase in IL-1β and IL-6, but not in TNF-α, during the early hematoma and inflammation phase in humans. Then, the levels of both IL-1 and IL-6 decrease while that of TNF-α increases [ 105 ]. The recruited neutrophils and M1 macrophages (until day 3) remove the damaged cells and tissue [ 108 ].…”

“…The recruited neutrophils and M1 macrophages (until day 3) remove the damaged cells and tissue [ 108 ]. During the resolution of acute inflammation, macrophages evolve to M2 phenotype, and the MSCs are recruited by a gradient of cytokines and chemoattractants, such as CXCL12 and MCP-1 (also called CCL2) [ 8 , 105 , 108 ]. The bone repair phase is initiated by the formation of the fibrocartilaginous callus.…”

“…However, Sarahrudi et al did not find any difference in the concentration of TGF-β1, in serum from patients with normal and impaired fracture healing, suggesting that further comparative studies must be performed to confirm TGF-β1 as a potential marker of non-union fractures [ 340 ]. In addition, Burska et al recently suggested that TGF-β2 and placenta growth factor might also be promising markers of human fracture healing [ 105 ].…”

An in vitro human-based fracture gap model – Mimicking the crosstalk between bone and immune cells

“…Since the bone marrow cavity is opened during the fracture, the bone marrow acts as a resource for chondro- and osteo-progenitor cells such as mesenchymal stromal cells (MSCs). Therefore, it can be hypothesized that osteogenic induction within the fracture gap is directly induced and controlled by signals from bone components in the vicinity of the fracture gap 11,12 . Hence, the crosstalk between immune cells from peripheral blood (after vessel rupture) and the bone marrow, and osteo-progenitor cells is essential and needs to be considered in preclinical studies 13 .…”