Abstract
Although controlled local inflammation is essential for adequate bone regeneration, several studies have shown that hyper-inflammatory conditions after major trauma are associated with impaired fracture healing. These hyper-inflammatory conditions include the trauma-induced systemic inflammatory response to major injury, open fractures, and significant injury to the surrounding soft tissues. The current literature suggests that increased or prolonged influx of neutrophils into the fracture hematoma may mediate impairment of bone regeneration after hyper-inflammatory conditions. The underlying mechanism remains unclear. We hypothesize that high neutrophil numbers inhibit synthesis of mineralized extracellular matrix (ECM) by bone marrow stromal cells (BMSCs). We therefore studied the effect of increasing concentrations of neutrophils on ECM synthesis by human BMSCs in vitro. Moreover, we determined how high neutrophil concentrations affect BMSC cell counts, as well as BMSC osteogenic activity determined by alkaline phosphatase (ALP) expression and ALP activity. Co-culture of BMSCs with neutrophils induced a 52% decrease in BMSC cell count (p < 0.01), a 64% decrease in the percentage of ALP+ cells (p < 0.001), a 28% decrease in total ALP activity (p < 0.01), and a significant decrease in the amount of mineralized ECM [38% decrease after 4 weeks (p < 0.05)]. Co-cultures with peripheral blood mononuclear cells and neutrophils within transwells did not induce a significant decrease in ALP activity. In conclusion, our data shows that a decreased amount of mineralized ECM became synthesized by BMSCs, when they were co-cultured with high neutrophil concentrations. Moreover, high neutrophil concentrations induced a decrease in BMSC cell counts and decreased ALP activity. Clarifying the underlying mechanism may contribute to development of therapies that augment bone regeneration or prevent impaired fracture healing after hyper-inflammatory conditions.
Highlights
Fracture healing starts with a controlled local inflammatory response, during which inflammatory cells infiltrate the fracture hematoma (FH) that surrounds the fracture [1]
Culture of bone marrow stromal cells (BMSCs) in 200 μl of Expansion medium (EM) for 7 days induced a 100% increase in BMSCs cell counts compared to BMSCs that were cultured in basic medium (BM) for 1 week (100%, n = 9, p < 0.001)
Co-culture of BMSCs with high neutrophil concentrations caused a 64% decrease in the percentage of alkaline phosphatase (ALP)+ cells within the entire BMSC population after 7 days of culture compared to monocultures of BMSCs (100%, n = 9, p < 0.001, Figure 2B)
Summary
Fracture healing starts with a controlled local inflammatory response, during which inflammatory cells infiltrate the fracture hematoma (FH) that surrounds the fracture [1]. It is commonly accepted that inflammatory cells initiate bone regeneration but are involved in the downstream processes of fracture healing [1, 2] This is illustrated by the finding that transplantation of the early FH into muscle. A controlled local inflammatory reaction seems essential for bone repair, several studies show that local and systemic hyper-inflammatory conditions are associated with impaired bone healing [1, 2]. These conditions include the trauma-induced [6] systemic immune response to major injury [7, 8], open fractures [9], and significant injury to the surrounding soft tissues [10]. In order to develop therapies that augment bone regeneration and/or prevent impairment of bone healing after hyper-inflammatory conditions, it is essential to understand how inflammatory cells influence the outcome of bone repair
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