Serum levels of bone turnover markers, bone metabolism proteins and cytokines.

<p>CTX-I levels are decreased in patients at 6 months follow-up when compared to healthy donors (p = 0.0268). DKK-1, IL-1 β, IL-6, IL-17A, IL-12p70, IL-23, TNF, MCP-1 and TGF-β are increased in patients at baseline when compared to healthy donors. After 6 months of therapy, follow-up patients had decreased levels of IL-17A, IL-23 and TGF-β when compared to their baseline. We have also observed that after therapy the levels of IL-1 β, IL-6, IL-12p70 and TNF were still significantly higher than healthy donors levels’. Each dot represents a sample. Line represents median. * vs Baseline, § vs Follow-up. DKK—dickkopf-related protein, CTX—carboxy-terminal collagen crosslinks, IL—interleukin, TNF—tumor necrosis factor, MCP—monocyte chemmotractant protein, TGF—transforming growth factor.</p

Gene expression profile of stimulated cells in culture for 21 days.

<p>All genes except CTSK are significantly decreased at day 1 in patients at follow-up when compared to healthy donors. At day 1 RANK expression in patients at baseline is also significantly reduced when compared to healthy donors (p = 0.0268). We found no differences between groups throughout the culture time except for Atp6v0d2 expression at day 21 in patients at baseline which is significantly reduced when compared both to healthy donors (p = 0.039) and patients at follow-up (p = 0.0234). Relative gene expression shown in Log scale. Dots in graphs represent median gene expression for each group at each time-point. d—day; CSF1R—gene encoding macrophage-colony stimulating factor (c-fms), RANK—gene encoding for receptor activator of nuclear factor-κβ, NFATc1—gene encoding for nuclear factor of activated T-cells, Atp6v0d2—gene encoding ATPase, H⁺ transporting, lysosomal V0 subunit D2, CTSK—gene encoding cathepsin K. p<0.05 is considered significant.</p

Osteoclast number is reduced in baseline patients, but bone resorption activity is increased after TNF-blocker exposure.

<p>A) Representative images of culture day 21 of cells stimulated with M-CSF, RANKL, dexamethasone and TGF-β and stained for TRAP. OC number increased throughout time and at culture day 21 baseline patients have significantly less osteoclasts than healthy donors (p = 0.0038). No differences were found in the number of nuclei per OC in any studied time of culture. B) Representative images of pit assay at culture day 21. Patients at follow-up had significantly higher number of pits and resorption area at culture day 21 when compared to their baseline (p = 0.0469 for both resorption pit number and percentage of resorbed area). Dots represent median counts for each group at each time-point and bars represent interquartile range [10–90]. d—day; OC—osteoclast; Scale bars 100μm, red arrows—osteoclasts, black arrows—resorption pits.</p

Description of the study population divided between the event of fracture and surgery.

<p>Values represent mean±standard deviation.</p><p>Comparisons between groups performed with ANOVA or chi-square tests.</p

Real time PCR primer sequences of the genes studied.

<p>B2M – β2-microglobulin; PMM1 - phosphomannomutase 1; IL1B – interleukin-1β; IL6 – interleukin-6; TNF – tumor necrosis factor; TGFB1 – transforming growth factor β1; BMP – Bone morphogenetic protein; IGF1 – insulin growth factor-1; FGF2 – fibroblast growth factor 2; PDGFB – platelet derived growth factor β; OPG – osteoprotegerin; RANK – receptor activator of nuclear factor κB; RANKL – RANK ligand; CBFA1/RUNX2 – core binding factor α1/runt-related transcription factor 2; OSX – osterix; ALP – alkaline phosphatase; SOST – sclerostin; TRAP – tartrate-resistant acid phosphatase; CTSK – cathepsin K; ITGB3 – subunit β3 of the integrin αvβ3; ATP - ATPase H⁺ transporter.</p

Comparison between the relative gene expression levels of patients submitted to hip replacement surgery due to low-energy fracture in relation to the days between fracture and surgery.

<p>Values represent median (Q1–Q3).</p><p>Comparisons between the 3 groups performed with Kruskall-Wallis H test.</p><p>*p-value for comparison between the 3 groups.</p><p>IL1B – interleukin-1β; IL6 – interleukin-6; TNF – tumor necrosis factor; TGFB1 – transforming growth factor β1; BMP – Bone morphogenetic protein; IGF1 – insulin growth factor-1; FGF2 – fibroblast growth factor 2; PDGFB – platelet derived growth factor β; OPG – osteoprotegerin; RANK – receptor activator of nuclear factor κB; RANKL – RANK ligand; CBFA1/RUNX2 – core binding factor α1/runt-related transcription factor 2; OSX – osterix; ALP – alkaline phosphatase; SOST – sclerostin; TRAP – tartrate-resistant acid phosphatase; CTSK – cathepsin K; ITGB3 – subunit β3 of the integrin αvβ3; ATP - ATPase H⁺ transporter.</p

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

<p>Each gene was normalized to the expression of the housekeeping genes <i>B2M</i> and <i>PMM1</i>. *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.</p

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

<p><i>RANK</i>, <i>RANKL</i> and <i>OPG</i> (<b>A</b>), osteoblast (<b>B</b>), osteocyte (<b>C</b>) and osteoclast-specific genes (<b>D</b>) were studied in the three study groups. Each gene was normalized to the expression of the housekeeping genes <i>B2M</i> and <i>PMM1</i>. *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.</p