Immunofluorescent labelling of cultured fibrocytes (n = 6 patients).

<p>Fibrocytes have been found to be double positive for Col1/LSP-1 and Col1/CD34. Our labelling here shows that mononuclear cells (MNCs) from dupuytren’s disease (DD) blood and DD fascia with or without serum amyloid P treatment were positive for both set of markers. We therefore confirmed that the cells in our experiments were fibrocytes and not resident fibroblasts. Magnification bar = 20 µm.</p

Presence of fibrocytes in dupuytren’s disease (DD) and control carpal tunnel (CT) blood and tissue confirmed through FACS and immunohistology.

<p>(<b>A–D</b>) The gating strategy used in identifying triple positive cells in CT and DD samples. The cells were labelled with antibodies against CD45RO, 25F9 and MRP8/14 cell surface markers. In a CD45RO vs. side scatter plot, CD45RO⁺ were selected and these cells were gated in a 25F9 vs. MRP8/14 plot. In the second plot, cells positive for both 25F9 and MRP8/14 were selected (along with total population as the gating was shown to be unnecessary as all 25F9⁺/MRP8/14⁺ cells were always CD45RO⁺) that are now considered to be triple positive (CD45RO⁺/25F9⁺/MRP8/14⁺). Cell viability was assessed separately through propidium iodide labelling. Cells were analyzed on Accuri C6 flow cytometer as explained in the materials and methods. (<b>E</b>) The fresh cells were obtained from the DD blood and tissue biopsies as explained in the methods. The cells were washed and passed through 70 µm nylon mesh filter before being labelled with antibodies as described above. Fibrocytes were highly expressed in freshly isolated MNCs from blood (1.8%) and in DD nodule (2.4%) compared to other tissue types. An unpaired two-tailed t-test was performed to check the statistical significance of the results. Error bars represent standard deviation. (<b>F</b>) H&E and immunofluorescence images of carpal tunnel (CT) fat and palmar fascia (n = 4 patients). CT fat and fascia are shown to be almost completely devoid of doubly positive collagen 1/CD34 cells. Red = Collagen I, Green = CD34, Blue = DAPI. CB = Collagen bundles. (<b>G</b>) H&E and immunofluorescence images of DD skin, cord and nodule (n = 4 patients). Cord typically shows linear arrangement of collagen bundles (LCB), while nodule here show circular arrangement of collagen fibers (CCB). DD cord and nodule showed the presence of clusters of doubly positive collagen 1/CD34 cells. Arrows points to the doubly positive cells that appeared as orange. Red = Collagen I, Green = CD34, Blue = DAPI, Orange = doubly positive cells. LCB = Linear collagen bundles; CCB = Circular collagen bundles. All scale bars: 100 µm.</p

Effects of Xiapex on dupuytren’s disease (DD) nodule.

<p>(<b>A</b>) 2×10⁴ cells derived from nodule were grown in 200 µl serum free media and treated with increasing concentrations of Xiapex (Collagenase <i>Clostridium histolyticum</i>) and collagenase A. Cell count was performed at day 8 as described in materials and methods. At concentration of 10 µg/ml and higher Xiapex inhibited differentiation of fibrocytes significantly (p<0.05) compared to collagenase A. Error bars represent standard deviation. (<b>B</b>) The brightfield images of fibrocyte cultures derived from DD nodule at various concentrations of Xiapex and collagenase A. Magnification bar = 50 µm.</p

Demographic Data of Dupuytren’s Disease Cases and Control Carpal Tunnel Subjects.

<p>Demographic Data of Dupuytren’s Disease Cases and Control Carpal Tunnel Subjects.</p

A flow chart depicting the study design.

<p>The flow chart here describes how the obtained tissue samples that were utilized for confirmation of the presence of fibrocytes in Dupuytren’s Disease.</p

Blood derived fibrocyte differentiation in the presence of serum amyloid p (SAP).

<p>(<b>A</b>) Fibrocyte counts at day 8 of <b>dupuytren’s disease</b> (DD) subjects (n = 10 patients) were higher than control carpal tunnel (CT) (n = 10 patients) for all concentrations of SAP. No significant difference was observed between CT and DD fibrocyte inhibition by SAP up to 2 µg/ml of SAP concentration. At 5 µg/ml SAP concentration virtually all CT MNCs were inhibited to differentiate into fibrocytes (p = 0.03). It took 10 µg/ml SAP to significantly reduce the number of fibrocytes for DD cultures. Error bars represent standard deviation. (<b>B</b>) H&E staining of day 8 fibrocytes (n = 5 patients), magnification 40x. Alongside of fibrocytes various other blood cells are visible, including monocytes, lymphocytes and a macrophage. (<b>C</b>) The bright field images of fibrocyte cultures at various concentrations of SAP. Magnification bar = 50 µm.</p