Summary of findings after de-tensioning of 3D tendon-constructs and TGF-β1 supplementation.

<p>The 3D tendon-construct under static tension without TGF-β1 is set as baseline, all other conditions are shown relative to the tensioned 3D tendon-construct (n = 5). A) Analysis of genes encoding for different matrix proteins: Collagen type I (COL1A1), collagen type III (COL3A1), collagen type XII (COL12A1) collagen type XIV (COL14A1) and fibronectin. B) Analysis of tendon phenotypic markers and tendon-related genes: tenomodulin (TNMD), scleraxis (SCX), Mohawk homeobox (MKX), fibromodulin (FBMD), decorin and GAPDH. Data presented on a logarithmic y scale as geometric means ± SEM with 3D tendon construct as baseline (n = 5). Significant 2-way ANOVA (tension*TGF-β1) main effects written above the graphs. For FBMD, * indicates significant effect of TGF-β1 for the individual groups in the post hoc analysis.</p

Release of Tensile Strain on Engineered Human Tendon Tissue Disturbs Cell Adhesions, Changes Matrix Architecture, and Induces an Inflammatory Phenotype

<div><p>Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro-inflammatory mediators and tendon phenotypic specific molecules, in an <i>in vitro</i> model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors showed a contrasting response with a clear drop in integrin subunit α₁₁ mRNA and protein expression, and an increase in α₂ integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules were upregulated. Stimulation with the cytokine TGF-β1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads to a decrease in phenotypical markers for tendon, while expression of pro-inflammatory mediators is induced.</p></div

Overview over the effect of de-tensioning on protein expression of different integrin subunits.

<p>A) Effect of de-tensioning on integrin sub-unit α5, B) Effect of de-tensioning on integrin sub-unit α11, C) Effect of de-tensioning on integrin sub-unit β1. The data are presented on a logarithmic y scale as geometric means ± SD (n = 3) where Tension Day 1 is baseline. Significant 2-way RM ANOVA (tension*time) main effects written above the graphs. Open triangles: 3D tendon-construct under tension, without TGF-β1 supplementation; open circles: 3D tendon-construct de-tensioned, without TGF-β1 supplementation. Below the graphs, representative western blots are shown (+/- tension).</p

Summary of mRNA expression of tendon-related genes as a response to changes in the spatial/mechanical environment and TGF-β1 supplementation.

<p>Summary of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086078#pone-0086078-g002" target="_blank">figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086078#pone-0086078-g003" target="_blank">3:</a> The effect of the intervention is compared to the gene expression of the respective target cultured for six days under static tension, the arrows (↑↓) indicate significant changes relative to the tensioned samples, or no statistical change (↔). Arrows in parenthesis indicate a tendency (p< 0.1). Tenomodulin (TNMD); Scleraxis (SCX); Mohawk homeobox (MKX); Collagen type I (COL1A1); collagen type III (COL3A1); collagen type XII (COL12A1); collagen type XIV (COL14A1); Fibronectin (FN); Decorin (DCN); Fibromodulin (FBMD); Transforming growth factor-β1 (TGFB1); Connective tissue growth factor (CCN2); Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).</p

Comparison between 2D monolayer and 3D tendon-constructs under static tension.

<p>The 3D tendon-construct is set as baseline, and mRNA expression of tendon cells grown on 2D monolayer is shown relative to the 3D tendon construct. Significant changes are indicated by *. Data presented on a logarithmic y scale baseline as geometric means ± SEM with 3D tendon-construct as baseline (n = 5).</p

Overview over the effect of de-tensioning and TGF-β1 supplementation on mRNA expression of tendon matrix genes and growth factors.

<p>Left panel (A, D, G, J): Effect of de-tensioning, mid panel (B, E, H, K): Effect of TGF-β1 on tensioned 3D tendon-constructs, right panel (C, F, I, L): Effect of TGF-β1 on de-tensioned 3D tendon-constructs. The data are presented on a logarithmic y scale as geometric means ± SD (n = 3) where Tension Day 1 is baseline. Significant 2-way RM ANOVA (tension*time) or (TGF-β1*time) main effects written above the graphs. Open triangles: 3D tendon-construct under tension, without TGF-β1 supplementation; open circles: 3D tendon-construct de-tensioned, without TGF-β1 supplementation; filled triangles: 3D tendon-construct under tension, with TGF-β1 supplementation; filled circles: 3D tendon-construct de-tensioned, with TGF-β1 supplementation.</p

Overview over the effect of de-tensioning on protein expression of integrin subunit α₂.

<p>The figure shows the western blot of one 3D tendon-construct over four time points shown(+/- tension).</p

Overview over the effect of de-tensioning and TGF-β1 supplementation on mRNA expression of phenotypic markers of tendon lineage.

<p>Left panel (A, D): Effect of de-tensioning, mid panel (B, E): Effect of TGF-β1 on tensioned 3D tendon-constructs, right panel (C, F): Effect of TGF-β1 on de-tensioned 3D tendon-constructs. The data are presented on a logarithmic y scale as geometric means ± SD (n = 3) where Tension Day 1 is baseline. Significant 2-way RM ANOVA (tension*time) or (TGF-β1*time) main effects written above the graphs. Open triangles: 3D tendon-construct under tension, without TGF-β1 supplementation; open circles: 3D tendon-construct de-tensioned, without TGF-β1 supplementation; filled triangles: 3D tendon-construct under tension, with TGF-β1 supplementation; filled circles: 3D tendon-construct de-tensioned, with TGF-β1 supplementation.</p

Overview of study design.

<p>Tendon cells on monolayer (“2D cells”) were harvested prior to seeding tendon cells into fibrin matrix to engineer human tendon-constructs. A) Culture condition under static tension, TGF-β1 supplementation to half of the samples at day 0 defined as the first time point at which a continuous linear matrix between the anchor points was formed; B) Tendon-constructs were cut to release tension at day 0, TGF-β1 supplementation to half of the samples.</p

Overview over primers used in the study.

<p>Large ribosomal protein P0 (RPLP0); Glyceraldehyde 3-phosphate dehydrogenase (GAPDH); Collagen type I, α1 chain (COL1A1); Collagen type III, α1 chain (COL3A1); ; Collagen type XII, α1 chain (COL12A1); Collagen type XIV, α1 chain (COL14A1); Scleraxis (SCX); Mohawk homeobox (MKX); Tenomodulin (TNMD) ; Fibronectin (FN); Fibromodulin (FBMD); Decorin (DCN); Integrin α₂ (ITGA2); Integrin α₅ (ITGA5); Integrin α₁₁ (ITGA11); Integrin β₁ (ITGB1); Transforming growth factor-β1 (TGFB1), Interleukin-1β (IL1B); Cyclooxygenase 1 (COX-1); Cyclooxygenase 2 (COX-2). Interleukin-6 (IL6); Matrix metalloproteinase (MMP).</p