Upregulation of inflammatory genes and downregulation of sclerostin gene expression are key elements in the early phase of fragility fracture healing

Abstract

Background: Fracture healing is orchestrated by a specific set of events that culminates in the repair of bone and reachievement of its biomechanical properties. The aim of our work was to study the sequence of gene expression events involved in inflammation and bone remodeling occurring in the early phases of callus formation in osteoporotic patients.

Methodology/principal findings: Fifty-six patients submitted to hip replacement surgery after a low-energy hip fracture were enrolled in this study. The patients were grouped according to the time interval between fracture and surgery: bone collected within 3 days after fracture (n = 13); between the 4(th) and 7(th) day (n = 33); and after one week from the fracture (n = 10). Inflammation- and bone metabolism-related genes were assessed at the fracture site. The expression of pro-inflammatory cytokines was increased in the first days after fracture. The genes responsible for bone formation and resorption were upregulated one week after fracture. The increase in RANKL expression occurred just before that, between the 4(th)-7(th) days after fracture. Sclerostin expression diminished during the first days after fracture.

Conclusions: The expression of inflammation-related genes, especially IL-6, is highest at the very first days after fracture but from day 4 onwards there is a shift towards bone remodeling genes, suggesting that the inflammatory phase triggers bone healing. We propose that an initial inflammatory stimulus and a decrease in sclerostin-related effects are the key components in fracture healing. In osteoporotic patients, cellular machinery seems to adequately react to the inflammatory stimulus, therefore local promotion of these events might constitute a promising medical intervention to accelerate fracture healing.

Figure 1. Relative expression of inflammation and growth factors genes grouped according to the time between the event of fracture and the surgery.

Each gene was normalized to the expression of the housekeeping genes B2M and PMM1. *p-value<0.05 for comparisons between the three groups. (Points represent median values). IL1B – interleukin-1β; IL6 – interleukin-6; TNF – tumor necrosis factor; IGF1 – insulin growth factor-1 ; FGF2 – fibroblast growth factor 2 ; PDGFB – platelet derived growth factor β; BMP – Bone morphogenetic protein; TGFB1 – transforming growth factor β1.

Figure 2. Relative expression of bone metabolism-related genes divided according to the time between the event of fracture and the surgery.

RANK, RANKL and OPG (A), osteoblast (B), osteocyte (C) and osteoclast-specific genes (D) were studied in the three study groups. Each gene was normalized to the expression of the housekeeping genes B2M and PMM1. *p-value<0.05 for comparisons between the three groups. (Points represent median values). RANK – receptor activator of nuclear factor κB; RANKL – RANK ligand; OPG – osteoprotegerin; CBFA1/RUNX2 – core binding factor α1/runt-related transcription factor 2; OSX – osterix; ALP – alkaline phosphatase; SOST – sclerostin; ITGB3 – subunit β3 of the integrin αvβ3; TRAP – tartrate-resistant acid phosphatase; ATP - ATPase H⁺ transporter; CTSK – cathepsin K.