Gene expression analysis of the pleiotropic effects of TGF-β1 in an in vitro model of flexor tendon healing.

Flexor tendon injuries are among the most challenging problems for hand surgeons and tissue engineers alike. Not only do flexor tendon injuries heal with poor mechanical strength, they can also form debilitating adhesions that may permanently impair hand function. While TGF-β1 is a necessary factor for regaining tendon strength, it is associated with scar and adhesion formation in the flexor tendons and other tissues as well as fibrotic diseases. The pleiotropic effects of TGF-β1 on tendon cells and tissue have not been characterized in detail. The goal of the present study was to identify the targets through which the effects of TGF-β1 on tendon healing could be altered. To accomplish this, we treated flexor tendon tenocytes cultured in pinned collagen gels with 1, 10 or 100 ng/mL of TGF-β1 and measured gel contraction and gene expression using RT-PCR up to 48 hours after treatment. Specifically, we studied the effects of TGF-β1 on the expression of collagens, fibronectin, proteoglycans, MMPs, MMP inhibitors, and the neotendon transcription factors, Scleraxis and Mohawk. Area contraction of the gels was not dose-dependent with the TGF-β1 concentrations tested. We observed dose-dependent downregulation of MMP-16 (MT3-MMP) and decorin, and upregulation of biglycan, collagen V, collagen XII, PAI-1, Scleraxis, and Mohawk by TGF-β1. Inter-gene analyses were also performed to further characterize the expression of ECM and MMP genes in the tenocyte-seeded collagen gels. These analyses illustrate that TGF-β1 tilts the balance of gene expression in favor of ECM synthesis rather than the matrix-remodeling MMPs, a possible means by which TGF-β1 promotes adhesion formation

rea contraction of the collagen gels as a functional measure of TGF-β1 activity.

<p>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).</p

TGF-β1 had little effect on MMP-2, MMP-3 and MMP-14 expression, but decreased expression of MMP-16.

<p>(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.</p

TGF-β1 did not affect TIMP-2 expression, but upregulated PAI-1.

<p>(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.</p

<i>In vitro</i> tendon healing model used to assess the effects of TGF-β1 on tenocyte gene expression.

<p>(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.</p

Primers used for RT-PCR analysis of gene expression.

<p>Primers used for RT-PCR analysis of gene expression.</p

TGF-β1 tilted the balance of ECM and MMP gene expression in favor of ECM.

<p>(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.</p

TGF-β1 highly upregulated Mohawk (<i>Mkx</i>) and Scleraxis (<i>Scx</i>), genes necessary for tendon development.

<p>(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.</p