Figure 1.
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.
Figure 2.
Comparison between 2D monolayer and 3D tendon-constructs under static tension.
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).
Figure 3.
Summary of findings after de-tensioning of 3D tendon-constructs and TGF-β1 supplementation.
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.
Figure 4.
Overview over the effect of de-tensioning and TGF-β1 supplementation on mRNA expression of collagen genes.
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.
Figure 5.
Overview over the effect of de-tensioning and TGF-β1 supplementation on mRNA expression of phenotypic markers of tendon lineage.
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.
Figure 6.
Overview over the effect of de-tensioning and TGF-β1 supplementation on mRNA expression of tendon matrix genes and growth factors.
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.
Figure 7.
Overview over the effect of de-tensioning and TGF-β1 supplementation on mRNA expression of different integrin subunits.
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. Integrin α2 (ITGA2); Integrin α5 (ITGA5); Integrin α11 (ITGA11); Integrin β1 (ITGB1). 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.
Figure 8.
Overview over the effect of de-tensioning on protein expression of different integrin subunits.
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).
Figure 9.
Overview over the effect of de-tensioning on protein expression of integrin subunit α2.
The figure shows the western blot of one 3D tendon-construct over four time points shown(+/- tension).
Figure 10.
Overview over the effect of de-tensioning and TGF-β1 supplementation on mRNA expression of pro-inflammatory mediators.
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.
Table 1.
Overview over primers used in the study.
Table 2.
Summary of mRNA expression of tendon-related genes as a response to changes in the spatial/mechanical environment and TGF-β1 supplementation.
Figure 11.
Visualizing of morphological changes to de-tensioning.
A) H&E staining of 3D tendon-construct harvested 24 hours under static tension. (B) 3D tendon-construct harvested 24 hours after the release of tension, (C) 3D tendon-construct harvested six days after the release of tension. D) Electron micrograph of cross-sectioned 3D tendon-construct harvested after 48 hours under static tension. The extracellular space shows regularly organized collagen fibrils. (E) 3D tendon-construct harvested 48 hours after release of tension: Besides the presence of aligned collagen fibrils, disorganized collagen fibrils become apparent (red arrows). (F) 3D tendon-construct harvested 48 hours after release of tension: Extracellular space with disorganized collagen fibrils in close proximity to tendon cells (red arrows).