INTRODUCTION: Flexor tendon healing continues to pose many challenges for hand surgeons and tissue engineers alike. The most common and debilitating adverse outcome of flexor tendon injuries is the formation of adhesions, which restrict the normal gliding motion of tendons through their synovial sheaths. Evidence exists that such adhesions are the result of excess scarring orchestrated primarily by the growth factor TGF-β1, which causes increased ECM synthesis and impaired remodeling. The purpose of the present study was to examine the effects of different doses of TGF-β1 on the contraction of pinned collagen gels, and the expression of ECM components as well as MMPs and their regulators by flexor tendon tenocytes in vitro. METHODS: Tissue Harvest & Cell Culture: All animals used in this study were cared for in compliance with regulations of the University Committee on Animal Research. Flexor digitorum longus tendons were obtained from five freshly sacrificed, 7 month old C57BL/6 mice. Specimens were stripped of surrounding tissue, washed in PBS and 1% Pen Strep, minced into 1 mm pieces, and trypsinized for 1 hr at room temperature under sterile conditions. The tendon fragments were then cultured in MEM α (GIBCO) supplemented with 20% FBS, 1% Pen Strep, and 6.5 µL/L 2-Mercaptoethanol. The tenocytes that emerged were serially passaged. At passage 5, aliquots of 7 million cells were cryopreserved at -80°C in 50% MEM α, 40% FBS and 10% DMSO. Cells were later thawed, plated, expanded and used at passage 7 for the experiment. Collagen Gel Contraction Model: FDL Tenocytes were suspended in neutral, isotonic collagen I (PureCol; Advanced BioMatrix) at a cell density of 700,000 cells/mL and cast into custom rectangular culture wells designed to fit into standard 6-well plates (gel volume of 0.45 mL). The cell-seeded collagen was gelled at 37°C for one hour, and scored with a sterile spatula to allow for contraction around two pins (Figure 1). After scoring, the gels were incubated overnight in 2 mL of MEM α supplemented with 1% FBS. The next day, gels were imaged at time 0, and their media was replaced with MEM α containing 1% FBS and 0, 1, 10, or 100 ng/mL of TGF-β1. Gels from each treatment group were imaged at 6, 24 and 48 hr to assess contraction, while other gels were frozen at -80°C for RNA analysis (n=3 per treatment per time point). RNA Analysis: Gels were thawed and vortexed briefly in 1 mL of Trizol and RNA was extracted using the TRIspin method [1]. Purified RNA was measured with a NanoDrop Spectrophotometer and 800 ng of RNA from each gel was reverse transcribed to cDNA (iScript cDNA Synthesis Kit; BioRad). A published sequence for 18s rRNA [2] along with validated primer sequences for fibronectin 1 (Fn1), collagen I (Col1a1), collagen III (Col3a1), PAI-1 (Serpine1), Mmp2, Mmp14, Timp2, and Scx from PrimerBank [3] were checked with NCBI's Primer-BLAST for specificity to the desired genes. Triplicate measurements of transcripts from each sample were performed with PerfeCTa SYBR Green FastMix (Quanta Biosciences), and the expression of target genes was normalized to 18s rRNA expression using the Pfaffl method [4]. Statistical Analysis: Gel area contraction and gene expression were compared with 2-way ANOVA and Bonferroni post-tests in Prism 4 (GraphPad). Statistical significance was defined as p < 0.05. RESULTS: Gel Contraction: Collagen gels treated with control media (MEM α + 1% FBS) contracted minimally over the course of 48 hr to only 93% of the initial gel area. Gels treated with 1, 10 and 100 ng/mL of TGF-β1, on the other hand, contracted to an average of 61%, 53% and 50% of their original area, respectively after 48 hr. There was a significant difference between TGF-β1 treated gels and controls after only 6 hr (p<.001) and between 1 ng/mL gels and gels treated with either 10 or 100 ng/mL at 24 (p<0.01) and 48 hr (p < 0.001). There was no statistical difference between gels treated with either 10 or 100 ng/mL. Gene Expression: Treatment with 10 and 100 ng/mL of TGF-β1 stimulated a 10-20-fold upregulation of Fn1 and Scx at 24 and 48 hr which was significantly different than controls where indicated by an asterisk in Figure 2 (top panels). The expression of Col1a1 and Col3a1 was also significantly increased 5-10 times by TGF-β1 in a dose-dependent manner at 24 and 48 hr (data not shown). Moderate (two-to four-fold) increases in Mmp2 (Figure 2, bottom-left), Mmp14 and Timp2 expression (data not shown) were observed with TGF-β1 treatment which reached significance at different time points, but no clear dose dependence was observed. All doses of TGF-β1 stimulated a significant increase in Serpine1 expression at 6 hr, which remained elevated only in the 100 ng/mL group at 24 and 48 hr (Figure 2, bottom-right). DISCUSSION: We investigated the effects of TGF-β1 on tenocyte-mediated collagen contraction and on the expression of tendon ECM proteins and their regulators. While all levels of TGF-β1 resulted in significant contraction compared to 1% FBS treated controls, 10 and 100 ng/mL TGF-β1 produced significantly greater contraction and notable differences in gene expression compared with 1 ng/mL TGF-β1. We also observed a significant, dose-dependent upregulation of Fn1, Col1a1, Col3a1 and Serpine1 in the TGF-β1 treated groups in agreement with what has been reported previously [5]. The high levels of Scx expression, a transcription factor key to tendon development, also agree with findings from an in vivo study of gene expression in early tendon healing [6]. The small changes in Mmp2, Mmp14 and Timp2 expression indicate that TGF-β1 may not strongly control expression of these ECM regulators, but does not rule out the possibility that TGF-β1 may regulate ECM turnover indirectly via other MMPs or intermediates such as PAI-1. This hypothesis is currently under investigation. SIGNIFICANCE: Elucidating the effects of TGF-β1 on the expression of ECM components and their regulators by tenocytes is an essential step towards understanding the mechanism by which adhesions are formed and key to the identification of treatments that may counteract the undesirable effects of TGF-β1 on flexor tendon healing.