Gene Expression Analysis of the Pleiotropic Effects of TGF-β1 in an In Vitro Model of Flexor Tendon Healing

Abstract

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.

Tendon development and musculoskeletal assembly: emerging roles for the extracellular matrix

“…Importantly, recent studies have shown that the expression and activity of MMPs are regulated by signals activated in response to mechanical forces, such as Itg and Tgfβ (Yu and Stamenkovic, 2000;Farhat et al, 2015). MMP misregulation also occurs upon tendon inflammation, and recent studies suggest that MMP inhibition can improve tendon repair (Bedi et al, 2010;Jelinsky et al, 2011;Farhat et al, 2012;Davis et al, 2013).…”

“…TGFβs are secreted bound to latent TGFβ-binding proteins (LTBPs), which form part of the large latency complex (LLC) in the ECM (Wipff et al, 2007;Maeda et al, 2011). They are also secreted along with latency-associated peptides (LAPs), which block association with TGFβ receptors, and along with other proteins of the LLC, they become incorporated into ECM via interactions between LTBPs and Fn, fibrillin or Dcn (Isogai et al, 2003;Rifkin, 2005;Farhat et al, 2012). In this manner, TGFβs are stored in the ECM and must be released from the LLC and LAPs in order to be 'activated' and available to interact with cognate receptors (Horiguchi et al, 2012).…”

Lactoferrin and parathyroid hormone are not harmful to primary tenocytes in vitro, but PDGF may be

“…Clinically, PDGF receptor expression is increased in diseased tendons, and is associated with hypercellularity 32 , and taken into context with our findings on the poor effects of PDGF on tendon-related genes, this suggests that PDGF may not be the most suitable factor for improving tendon healing outcomes. IGF-1 and TGF-β are both present within the tendon matrix 18 , and in vitro have been shown to be involved in tendon cell growth, collagen production and matrix remodelling [19][20][21][22][23][24][25] . Developmental studies suggest a key role for TGFβ1 in tendon development 33 , and inhibiting TGFβ1 has generally resulted in poor healing outcomes in in vivo tendon defect models 34 .…”

“…These were chosen as PDGF is among the most widely studied tendon factors, and IGF-1 and TGF-β are both present within the tendon matrix. Furthermore, all are upregulated during tendon healing, and are known to enhance tendon cell growth, collagen production and matrix remodelling, in vitro [18][19][20][21][22][23][24][25][26] . Furthermore, we have focussed on not only classical tendon-related outputs, such as cell proliferation and collagen production, we have also assessed the tenocyte gene expression profile, assessing genes important in tenocyte, chondrocyte and osteoblast biology.…”

Delivery of bone morphogenetic protein-2 and substance P using graphene oxide for bone regeneration

“…Collagen gel was prepared according to Farhat's protocol. 37 Type I collagen (Sigma-Aldrich) was dissolved in water with acetic acid resulting in a pH of 3.0. The collagen solution was neutralized with PBS and NaOH to a pH of approximately 7.4.…”