Surgical specimen were collected in strict compliance with the Institute's chief pathologist and the ethics committee for research involving human subjects at UWO. Briefly, diseased fascia (cords) and adjacent, uninvolved (control) palmar fascia tissue was collected and divided in two portions for protein analysis (immediately stored -80°C) and primary cell cultures. For explant cultures, fascia specimen were finely minced and placed in starter media consisting of α-MEM (Gibco, Invitrogen Corporation, Grand Island, NY) containing 20% fetal bovine serum (FBS, Clontech Laboratories, Palo Alto, CA) and antibiotics (Penicillin G + streptomycin sulfate, Gibco, Invitrogen Corporation, Grand Island, NY). Established primary cell culture lines were maintained in α-MEM containing 10% FBS and antibiotics. Culture flasks were incubated at 37°C in a humidified chambers with 5% CO₂. Medium was changed every 4–5 days and the cells were sub-cultured using 0.05% Trypsin-EDTA (Gibco, Invitrogen Corporation, Grand Island, NY) when confluent.

Collagen contraction assays were carried out using disease (D) and control (C) primary cell cultures established from lesional and adjacent normal (uninvolved) fascia from the same patient (i.e. patient-matched), respectively. Primary cultures (passages 4–6) were grown as three dimensional Fibroblast Populated Collagen Lattices, or FPCL (Fig. 3). Collagen lattices were prepared by mixing cells with a neutralized solution of collagen type I (8 parts Vitrogen100 collagen type I, 2.9 mg/ml, Collagen Corp, Santa Clara, CA, USA + 1 part 10x α-MEM + 1 part HEPES buffer, pH 9). Final collagen and cell concentrations for the FPCL were 2.0 mg/ml and 2 × 10⁵ cells/ml of matrix, respectively. The cell-collagen mixture was then aliquoted into 24 well culture dishes (0.5 ml/well) that were pre-treated with a PBS + 2% BSA solution. Once polymerized (1 hr, 37°C) α-MEM + 10% fetal calf serum (FCS) was added atop FPCLs in each well. After 2 days of incubation, the attached FPCL were mechanically released from the sides of the culture plates. Digital images of the contracting FPCL were captured at various time points over 5 days using a conventional flatbed scanner. Collagen lattice areas were then measured using NIH imaging software.

FPCL were harvested at various post-mechanical released time points, homogenized and protein extracts prepared using a modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40, 150 mM NaCl, 1 mM EDTA) supplemented with a cocktail of protease inhibitors (1 mM PMSF, 1 μg/ml of aprotinin, leupeptin, pepstatin) and phosphatase inhibitors (1 mM Na₃VO₄, 1 mM NaF) (Sigma, St. Louis MO, USA). The resulting FPCL homogenate was then centrifuged (15 min, 4°C 12,000 × g) to remove cell debris. Cells numbers were quantified using an in vitro toxicology assay kit based on lactic dehydrogenase (LDH) levels (Sigma, St. Louis MO, USA), according to the manufacturers instructions. Equivalent cellular protein levels were then analyzed by SDS-PAGE (12%) and subsequently transferred to polyvinylidene difuoride (PVDF) membranes (BioRad, Hercules CA, USA). Membranes were blocked overnight (4°C) with a PBS solution containing 0.1% Tween 20 (PBST) and 5% fat free skim milk, washed 2x in PBST, and then sequentially probed with anti-β-catenin (1:750, Transduction Laboratories, Lexington, KY, USA), anti-Fn (clone IST-4, 1:500, Sigma, St. Louis MO, USA), and anti-heat shock protein 47 (Hsp47) antibodies (1:500, StressGen, Victoria BC, Canada). After a brief series of PBS washes membranes were then incubated with the horseradish peroxidase (HRP) conjugated 2° antibodies (1:5000, Jackson ImmunoResearch, West Grove PA, USA) for 45 min. at room temperature (RT). Antibody-specific bands were visualized using enhanced chemiluminescence (ECL) reagents and Kodak XLS film (Rochester NY, USA). Antibody-specific bands for β-catenin, fibronectin and Hsp47 were quantified using NIH Imaging software. Both β-catenin and fibronectin levels were normalized to control Hsp47 levels by calculating the appropriate ratio values (i.e. β-catenin:Hsp47, and Fn:Hsp47). The plotted bar graph values therefore represent the mean ratio intensity ± SEM per time point. For attached-matrix experiments, harvested FPCLs were treated as described above except that they were not mechanically released from the sides of the dishes throughout the 5 day incubation period.

Individual lattices were briefly washed in PBS, fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington PA, USA) for 1 hr at room temperature (RT), permeabilized in PBS + 0.5%Tween (PBST) for 15 min at RT, and then blocked overnight at 4°C with PBS + 2% donkey serum (Jackson ImmunoResearch, West Grove PA, USA). Lattices were then incubated with the nucleic acid stain DAPI (4',6-diamidino-2-phenylindole, dihydrochloride, Molecular Probes, Eugene ON, USA) for 20 min at RT, and then stained for 20 min at RT with phalloidin-Alexa 488 (Molecular Probes, Eugene ON, USA). The lattices were then briefly washed in PBS and mounted onto glass slides using aqueous DAKO faramount mounting media (DAKO Diagnostics, Carpinteria CA, USA). Digital images were acquired on a Nikon eclipse TE-200 inverted fluorescent microscope using a Photometrics series 300 cooled CCD camera, and deconvolved using softWoRx (v 2.5) software (Applied Precision Inc., Issaquah WA, USA).

FPCL cultures of DD primary cell lines not only provides a functional assay to quantify cell-mediated collagen contraction events, but also an 'in vivo-like' environment (i.e. three-dimensional collagen-rich matrix environment) to study cell-matrix interactions. Primary cell lines established from patient-matched disease fascia and adjacent uninvolved normal fascia tissue (n = 5 patient) were cultured as 'stressed-matrices' (Fig. 3). Following 2 days of attached-culture FPCLs were mechanically released from the sides of the culture dishes and digital images of the contracting gels collected over a 5 day incubation period. As shown in Fig. 4, area measurements of the contracting FPCL for all five patient-matched primary cell lines show that the disease cell cultures contracted collagen significantly more between 30 min. and 5 days compared to control FPCLs (*P < 0.05, student T-test). The differences in contraction rates is consistent with a number of earlier FPCL studies that demonstrated enhanced contractile activity of fibroblasts derived from DD lesions 474849. However, these studies utilized carpal tunnel fibroblasts as control cells and often used a floating-matrix rather than stressed-matrix contraction model to quantify cell-mediated collagen contraction, thus making it difficult to directly compare results.

Stressed-collagen lattices stained with phalloidin-Alexa 488, a fluorescent probe that binds filamentous actin, revealed more prominent stress fiber networks in the disease FPCLs compared to control FPCLs (Fig. 5), suggesting that the disease cells may be more inherently responsive to factors that promote collagen contraction. Although the exact nature of the cell signalling networks responsible for the enhanced contractile activity of the disease cells is unclear (e.g. cell-ECM interactions, growth factor pathways), the three-dimensional collagen matrix environment appears to differentially regulate the activity of the control and disease cells.

Since β-catenin is aberrantly expressed in vivo and in vitro (FPCL cultures) in DD 13, we examined its levels in relation to changes in mechanical stress (stressed- or attached-matrices). Fibronectin (Fn) levels were also examined since it plays an important role in cell-collagen interactions 435051, and the regulation and function of β-catenin 5253. During the contraction of stressed-FPCLs β-catenin levels transiently increased in disease cells, peaking at 1 hr post-mechanical release (R1, Dis/ctrl ratio = 3.6) and decreasing thereafter to levels equivalent to, or below those seen in control FPCLs, while in control matrix cultures β-catenin levels did not significantly change throughout the incubation period (Fig. 6). By contrast, Fn levels in disease FPCLs (R0, Dis/ctrl ratio = 2.7) steadily decreased upon mechanical release of the matrices, while Fn levels in control lattices showed a slight increase during contraction (Fig. 6).

While the observed decline in Fn levels in the disease FPCLs is consistent with previous studies that show disassemble of cellular Fn fibrils upon loss of isometric tension 5455, the lack of change in Fn levels in the contracting control FPCLs is unclear. Nevertheless it is conceivable that the differences between the disease and normal fibroblasts with respect to stress-induced changes in Fn may simply reflect the disease-specific nature of these cells since Fn is a gene target of the β-catenin/Lef-Tcf transcriptional complex 52. Considering that Fn can stimulate β-catenin nuclear accumulation via ILK (integrin-linked kinase) 56, and ILK itself can up-regulate Fn matrix assembly 57, it is possible that both β-catenin and Fn may share a common pathway within these cells. Although the mechanism responsible for the tension-dependent decrease in β-catenin is not clear it may also be related to the non-proliferative state of relaxed or floating FPCL cultures (Fig. 3) 58.

To further explore β-catenin and its relation to mechanical stress, we also analyzed β-catenin levels within attached-matrix (see Fig. 3) FPCL cultures. As shown in Fig. 7, western analysis of attached-matrices showed an accumulation of β-catenin in the disease cells over time, relative to control Hsp47 levels, while β-catenin levels remain relatively unchanged in the control cells. Although Fn levels in both control and disease cultures increased over the incubation period, Fn levels accumulated much faster within the disease FPCLs compared to control FPCL cultures (Fig. 7). Although the exact mechanism responsible for the tension stimulated accumulation of β-catenin is not clear it may be related to proliferative nature of attached-matrix cultures 58.

Given that Fn levels increase much earlier in the disease FPCL cultures, and that Fn matrix can regulate β-catenin levels, it is possible that disease cell cultures develop a peri-cellular Fn matrix (fibronexus) much earlier, and perhaps to a much greater extent, than control cells. Since previous studies have shown that the formation of filamentous actin (F-actin) networks parallels Fn matrix assembly 55, we probed FPCLs for F-actin expression using phalloidin-Alexa 488. Immunocytochemistry (ICC) analysis shows that the disease cells form extensive stress-fibres Fig. 5 and attach and spread within the collagen matrix much earlier than control cells Fig. 8, suggesting that the disease cells form a Fn matrix much earlier than control cells within attached-matrix cultures. While it is not clear whether β-catenin up regulates cellular Fn production, and/or cellular Fn stimulates increased β-catenin stability, it does appear that this altered metabolic state of the disease cells promotes cell-collagen attachment and cell-mediated collagen contraction. The build-up of isometric tension within the FPCL cultures likely accelerated this process since isometric tension can directly modulate cell signalling events important to fibroblast contractility 59, as well as stimulate the expression of contractile markers such as Fn and α-SM actin in vivo 60. Moreover, the well recognized role that integrins (cell-ECM receptors) play in helping to generate isometric tension and Fn matrix assembly 5161, suggests that integrin-mediated signalling factors, like ILK, may provide a possible mechano-chemical pathway linking isometric tension with changes in the β-catenin and Fn levels. Nevertheless, the possible relationship between β-catenin, Fn, ILK and other signalling factors remains to be examined.