Figure 1.
In vitro tendon healing model used to assess the effects of TGF-β1 on tenocyte gene expression.
(A) For each experiment, tenocyte-seeded collagen was cast into the collagen gel well of a custom culture construct between two screws. After gelation, 2 mL of media was added to the culture media well. (B) Tenocyte-seeded collagen gels treated with TGF-β1 contracted over 48 hours and aligned themselves between two screws, forming a tissue that grossly resembled tendon.
Figure 2.
rea contraction of the collagen gels as a functional measure of TGF-β1 activity.
Digital images of tenocyte-seeded collagen gels treated with control media (containing 1% FBS and 1% Pen Strep) supplemented with 0, 1, 10 or 100 ng/mL of TGF-β1 were analyzed using ImageJ. The area ratio (gel area divided by the area at 0 hours) was determined at 0, 6, 24 and 48 hours after treatment to assess contraction. Gels treated with 1–100 ng/mL of TGF-β1 contracted significantly more than controls after only 6 hours (p<0.001). No differences in area ratio were observed between the three doses of TGF-β1 at any time except in gels treated with 1 vs. 100 ng/mL at 48 hours (p<0.05). N = 6 gels per treatment per time point. Error bars represent the standard error of the mean (SEM).
Figure 3.
TGF-β1 increased the expression of fibronectin and collagen genes.
(A–E) The mean expression (± SEM) of individual fibronectin and collagen genes in tenocyte-seeded collagen gels after treatment with control media supplemented with 0–100 ng/mL of TGF-β1 over 48 hours was assessed with RT-PCR. Only collagens I, III and XII were upregulated significantly by the lowest dose, 1 ng/mL of TGF-β1. On the other hand, all of the ECM genes were upregulated by 10 and 100 ng/mL doses of TGF-β1 at 24 or 48 hours. The highest dose, 100 ng/mL, had the longest-lasting effects and caused the greatest upregulation of all genes at 48 hours. N = 5−6 gels per treatment per time point. *p<0.05 vs. control media, •p<0.05 vs. 1 ng/mL TGF-β1, +p<0.05 vs. 10 ng/mL TGF-β1. (F) Inter-gene analysis of fibronectin and collagen expression (± SEM) before (0 hours) and 48 hours after treatment with 0–100 ng/mL TGF-β1 normalized to 0 hours. The expression of all four collagens combined (red) was approximately equal to the expression of fibronectin (blue) at each time point and treatment condition. (G) Inter-gene analysis of the expression levels of individual collagen isoforms (± SEM) normalized to 0 hours. Collagen III was the most highly expressed isoform in tenocytes cultured in collagen gels before and after TGF-β1 treatment. Collagen I was the next most expressed isoform. The other two isoforms, collagen V and XII, each represented only 4–12% of the total collagen transcripts analyzed, but their expression increased substantially after treatment with 100 ng/mL of TGF-β1 to levels similar to the expression of collagens I and III before treatment (0 hours).
Figure 4.
TGF-β1 increased the expression of biglycan and decreased the expression of decorin.
(A–C) The mean expression (± SEM) of proteoglycan genes coding for biglycan, decorin, and lumican was evaluated in tenocyte-seeded collagen gels after treatment with control media or 1–100 ng/mL of TGF-β1 over 48 hours. Biglycan expression was significantly increased in the 10 and 100 ng/mL treatment groups at 24 and 48 hours (p<0.05, Panel A). Decorin expression, on the other hand, increased 14-fold in the control and 1 ng/mL treatment groups, but this increase was suppressed by 10 and 100 ng/mL of TGF-β1 (p<0.05, Panel B). Lumican expression was significantly increased with 1 and 10 ng/mL TGF-β1 (p = 0.001), but not 100 ng/mL TGF-β1 (Panel C). N = 5−6 gels per treatment per time point. *p<0.05 vs. control media, •p<0.05 vs. 1 ng/mL TGF-β1. (D) Inter-gene analysis of the proteoglycans revealed that biglycan expression was the highest at all treatments and time points (82–98%), followed by decorin (2–17%) and lumican (<2%). (E) The relative transcript levels of the proteoglycans, fibronectin, and collagen were also determined. At each treatment and time point, proteoglycans (red) were expressed most highly, followed by fibronectin (white) and collagen (blue). All categories of ECM genes were upregulated in a dose-dependent manner by 1–100 ng/mL of TGF-β1 at 48 hours.
Figure 5.
TGF-β1 had little effect on MMP-2, MMP-3 and MMP-14 expression, but decreased expression of MMP-16.
(A–D) The mean expression (± SEM) of MMP genes were evaluated in tenocyte-seeded collagen gels after treatment with control media or 1–100 ng/mL of TGF-β1 over 48 hours. MMP-2 (A) and MMP-14 (C) increased 1.5- to 4-fold over 48 hours, but were not significantly affected by treatment with 1–100 ng/mL of TGF-β1. While TGF-β1 treated gels expressed about twice as much MMP-3 compared to control gels at 48 hours, only the 1 ng/mL TGF-β1 treatment group reached significance (p<0.01, Panel B). MMP-16 expression, on the other hand, increased 4- to 5-fold at 24 and 48 hours in the control and 1 ng/mL groups, but this increase was significantly reduced in the 10 and 100 ng/mL TGF-β1 treated gels (p<0.01, Panel D). N = 5−6 gels per treatment per time point. *p<0.01 vs. control media, •p<0.01 vs. 1 ng/mL TGF-β1. (E) Inter-gene analysis of the expression of the MMPs before and 48 hours after treatment with TGF-β1. At 0 hours, MMP-14 expression was roughly equal to the other MMPs combined; however, after 48 hours, the levels of MMP-2 and MMP-3 increased in all groups regardless of the presence of TGF-β1. MMP-16 constituted the smallest portion of MMP expression in all treatment groups and time points.
Figure 6.
TGF-β1 did not affect TIMP-2 expression, but upregulated PAI-1.
(A) PAI-1 responded to all three doses of TGF-β1 with significant upregulation at as early as 6 hours (p<0.05). However, only the 100 ng/mL dose of TGF-β1 appeared to have sustained effects on PAI-1 expression at 24 and 48 hours. (B) TIMP-2 expression increased about 2-fold in all treatment groups over 48 hours and was not significantly affected by TGF-β1 at any concentration or time point tested. N = 5−6 gels per treatment per time point. *p<0.05 vs. control media, •p<0.001 vs. 1 ng/mL TGF-β1, +p<0.001 vs. 10 ng/mL TGF-β1.
Figure 7.
TGF-β1 highly upregulated Mohawk (Mkx) and Scleraxis (Scx), genes necessary for tendon development.
(A) Mohawk was significantly upregulated by 10 and 100 ng/mL TGF-β1 at 48 hours. (B) Scleraxis was upregulated by 100 ng/mL at 6 hours, and 10 and 100 ng/mL at 24 and 48 hours. N = 5−6 gels per treatment per time point. *p<0.05 vs. control media, •p<0.001 vs. 1 ng/mL TGF-β1.
Figure 8.
TGF-β1 tilted the balance of ECM and MMP gene expression in favor of ECM.
(A) A comparison of the total ECM (includes fibronectin, collagen and proteoglycan genes) vs. total MMP expression (includes MMP-2, -3, -14 and -16 genes) illustrates that TGF-β1 caused dose-dependent increases in overall ECM transcription, but not MMP expression at 48 hours. (B) The ratio of ECM to MMP expression was calculated for each treatment and time point. Gels treated with 10 or 100 ng/mL of TGF-β1 had significantly higher ECM/MMP ratio at 24 hours compared to controls. Gels treated with 100 ng/mL of TGF-β1 also had a significantly higher ECM/MMP ratio at 48 hours. The 1 ng/mL treatment group, however, was not sufficient to alter the ECM/MMP ratio at any time point. *p<0.05 vs. control media, •p<0.01 vs. 1 ng/mL TGF-β1.
Table 1.
Primers used for RT-PCR analysis of gene expression.