The authors have declared that no competing interests exist.
Conceived and designed the experiments: YMF HAA. Performed the experiments: YMF AAA TC SCJ. Analyzed the data: YMF AAA TC SCJ EMS RJO HAA. Contributed reagents/materials/analysis tools: TC SCJ. Wrote the paper: YMF AAA TC SCJ EMS RJO HAA.
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.
Each year, millions of Americans injure their hands in the workplace, home and elsewhere resulting in significant morbidity and thousands of lost workdays
Evidence exists that flexor tendon adhesions
The ultimate goal of flexor tendon repair is to restore the mechanics of the tendon to that of its uninjured state. The composition and organization of the extracellular matrix (ECM) of uninjured tendon is what defines its mechanical strength and behavior. The major component of tendon ECM is collagen, a rope-like protein which forms several levels of hierarchical structures called fibrils, fibers and fascicles (reviewed in
The condition of the ECM in developing, normal, and healing tissue is both a function of ECM production and degradation. Matrix metalloproteinases (MMPs) are the class of proteinases that are best known for their ability to degrade various ECM components, including collagen, fibronectin, and proteoglycans
It is also theorized that regenerative, scarless healing of force transmitting tendons would require the activation of Scleraxis (
Collagen hydrogels are a desirable model of tendon healing, because collagen is the primary constituent of tendon
For the present experiments, we designed silicone constructs that fit into 6-well plates and accommodate approximately 450 µL of cell-seeded collagen I, which is gelled between two polymer screws (
(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.
Gel contraction at 0, 6, 24 and 48 hours after treatment was calculated based on surface area in order to evaluate the degree of cellular remodeling elicited by the three doses of TGF-β1 (
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).
Real-Time Polymerase Chain Reaction (RT-PCR) was performed on tenocyte-seeded collagen gels at 0, 6, 24 and 48 hours after treatment to evaluate the effects of TGF-β1 on gene expression. The gene expression of the extracellular matrix proteins, fibronectin and collagen types I, III, V and XII increased in gels treated with TGF-β1 (
(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).
A novel inter-gene analysis was performed to compare the expression of fibronectin and collagen types I, III, V and XII in tenocyte-seeded collagen gels before (0 hours) and 48 hours after treatment with TGF-β1. First, the total expression of all four collagens combined (
We also compared the relative transcript levels of the four collagen types analyzed at 0 and 48 hours for each treatment group (
The expression of the small leucine-rich proteoglycans (SLRPs), biglycan, decorin, and lumican, in tenocyte-seeded collagen gels treated with control media or 1–100 ng/mL of TGF-β1 was also assessed with RT-PCR (
(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.
An inter-gene analysis was used to assess the relative expression of biglycan, decorin and lumican in the tenocyte-seeded collagen gels before and 48 hours after treatment with TGF-β1 (
The relative transcript levels of proteoglycans, fibronectin and collagen were similarly evaluated (
Although TGF-β1 had only a small effect on the expression of MMP-2, MMP-3, and MMP-14, it resulted in a marked dose-dependent reduction in the gene expression of MMP-16 in a dose-dependent manner (
(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.
An inter-gene analysis of the relative levels of MMPs was performed (
PAI-1 expression was upregulated approximately 2-fold after only 6 hours by all three doses of TGF-β1 (p<0.05 -
(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.
Both neotendon transcription factors, Scleraxis (
(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.
An inter-gene analysis was performed to compare the total number of ECM transcripts (i.e. of fibronectin, collagen and proteoglycan genes) with the total number of MMP transcripts (MMP-2, -3, -14 and -16). This analysis showed that higher doses of TGF-β1 caused major increases in ECM expression (red), but not MMP expression (blue) at 48 hours (
(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.
The ratio of expression of ECM to MMP genes was also determined for all treatments and time points (
Using an
As fibronectin, collagen I, and collagen III are the main components of scar tissue and adhesions, it was expected that TGF-β1 would cause large increases in the expression of these genes. However, we did not expect TGF-β1 to have such a profound effect on collagens V and XII, which are normally associated with the maturation of collagen fibrils during normal tendon development
We also examined the effects of TGF-β1 on the expression of biglycan, decorin, and lumican, proteoglycans which play an important role in the development of tendon and regulation of collagen fibrillogenesis (reviewed in
Given the important role MMPs play in the turnover of ECM, we hypothesized that TGF-β1 may promote adhesion formation by inhibiting expression of MMPs or upregulating MMP activity modulators (PAI-1, TIMP-2). In terms of MMP expression, TGF-β1 did not inhibit the transcription of MMP-2, MMP-3 or MMP-14. Interestingly, their expression was upregulated over the course of the experiment in a time-dependent manner. This finding is consistent with the observation that fibroblast-mediated collagen gel contraction is MMP-2 and MMP-3 dependent
MMP-16 expression was downregulated by TGF-β1, a novel finding in this experiment. MMP-16 is a membrane-bound MMP which activates other MMPs and promotes collagen fibril formation during tendon development
Finally, the inter-gene analysis of ECM genes strikingly revealed that the tenocytes seeded in collagen I gels produced very high levels of fibronectin (
One important limitation of this study was that tenocytes were subjected to the constantly changing microenvironment of contracting collagen gels. While the changing microenvironment is a potential confounding variable, the data suggests that it did not have a strong effect on gene expression. Firstly, gels treated with 1 ng/mL of TGF-β1 contracted to a similar extent as gels treated with 10 or 100 ng/mL at all time points. The contraction data therefore suggest that gels treated with 1–100 ng/mL TGF-β1 had similar microenvironments throughout the experiment, and that their microenvironments may have differed from the control gels as the gels were remodeled. Therefore, if differences in the microenvironment had a large effect on gene expression, this would cause gels treated with 1 ng/mL to more closely resemble the 10 and 100 ng/mL treatment groups, rather than the control group (as was the case in terms of gel contraction). However, in almost every gene analyzed, the 1 ng/mL gels most closely resembled the control gels at 24 and 48 hours when contraction was most pronounced, suggesting that changes in the microenvironment of the gels did not have a large effect on gene expression throughout duration of the experiment.
There are several other limitations to this study. One was that the tendon-derived cells, which we have thus far referred to as “tenocytes”, are likely a mixed population of cells including epitenon and endotenon fibroblasts, tendon progenitor/stem cells (TSCs), and vascular-associated cells
As our goal was to evaluate the effects of TGF-β1 on gene expression, an important limitation of this study was that protein levels beyond the transcription level were not assessed. We also did not evaluate the signaling pathways involved with TGF-β1 signal transduction such as the Smad
In conclusion, flexor tendon healing is a complex clinical challenge that requires a detailed understanding of the numerous factors that are involved in complications associated with tendon repairs; namely, inferior repair strength and the formation of debilitating adhesions. Our analysis of TGF-β1’s effects on flexor tendon tenocytes not only provided insights into the positive effects of TGF-β1 on tendon regeneration, it also confirmed the unavoidable fibrotic effects of this factor in terms of tilting the balance of ECM and MMP expression in favor of the former, upregulating the expression of the MMP activity inhibitor, PAI-1, and downregulating the expression of MMP-16. Future studies are warranted to functionally define the role of MMPs in this model and further understand the implications of our findings to the problems associated with flexor tendon healing.
All animals (C57BL/6 mice) used in this study were cared for in accordance with an animal use and care protocol approved by the University Committee on Animal Research (UCAR) of the University of Rochester. Mice were used solely for tendon harvest to isolate tenocytes from flexor tendons of the hind paws. In brief, mice were sacrificed in approved CO2 euthanasia chambers and death was verified using cervical dislocation. No live mice were used in this study.
Flexor digitorum longus tendons were obtained from the hind limbs of five freshly sacrificed, 7 month old C57BL/6 mice. Specimens were stripped of surrounding tissue, washed in DPBS (Gibco, #14190) and 1% Pen Strep (Gibco, #15140), minced into 1 mm pieces, and trypsinized for 1 hour at room temperature under sterile conditions. The tendon fragments were then cultured in MEM α (Gibco, #12561) supplemented with 20% FBS (Sigma-Aldrich, #F6178), 1% Pen Strep, and 6.5 µL/L of 2-Mercaptoethanol (Sigma-Aldrich, #M7522). The cells that emerged were serially passaged five times and then aliquots of 7 million cells were cryopreserved at −80°C in 50% MEM α, 40% FBS and 10% DMSO (Sigma-Aldrich, #D2650). The tendon cells were later thawed, plated, expanded and used at passage 7 for each of the experiments. As the cells at passage five overwhelmingly exhibited an elongated, fibroblast-like morphology, they were termed “tenocytes”
The day before each experiment, near-confluent tenocytes were trypsinized for 10 minutes in 0.25% Trypsin-EDTA (Gibco, #25200) in a humidified incubator (5% CO2, 37°C). Cells were then washed in culture media, centrifuged, strained to ensure a single cell suspension, and counted using a hemocytometer. The cells were then pelleted, resuspended in control media (MEM α supplemented with 1% FBS and 1% Pen Strep), and mixed with an isotonic, neutral collagen I solution (Advanced BioMatrix, #5005-B) at a volume ratio of 1∶19 to achieve a final cell density of 7×105 cells/mL and collagen concentration of 2.3 mg/mL. The cell-seeded collagen was then cast into custom-made silicone constructs (
After gelation, the edges of the collagen gels were separated from the sides of the silicone construct with a sterile spatula, and 2 mL of control media was added to the media well of each construct (
Tenocyte-seeded collagen gels treated with control media supplemented with 0, 1, 10 or 100 ng/mL of TGF-β1 were collected at 0, 6, 24 and 48 hours and frozen at −80°C for RNA purification and reverse-transcription. Within two weeks of each experiment, gels were thawed and vortexed briefly in 1 mL of TRIzol (Ambion, #15596) and RNA was extracted using a modified version of the TRIspin method
Immediately after purification, the quantity and purity of each sample was measured with a spectrophotometer (NanoDrop 1000). Subsequently, 800–1000 ng of RNA from each gel was reverse-transcribed to cDNA using the iScript cDNA Synthesis Kit (BioRad, #170-8891), and samples were stored at −20°C for gene expression analysis with RT-PCR.
Validated primer sequences for all of the genes analyzed were obtained from PrimerBank
Gene | Forward Primer (5′-3′) | Reverse Primer (5′-3′) | Amplicon Length |
Biglycan |
|
|
112 |
Collagen, type I |
|
|
103 |
Collagen, type III |
|
|
154 |
Collagen, type V |
|
|
97 |
Collagen, type XII |
|
|
111 |
Decorin |
|
|
81 |
Eukaryotic translation elongation factor 1 alpha 1 |
|
|
70 |
Fibronectin ( |
|
|
105 |
Lumican |
|
|
147 |
Mohawk ( |
|
|
73 |
Matrix Metalloproteinase-2 |
|
|
171 |
Matrix Metalloproteinase-3 |
|
|
192 |
Matrix Metalloproteinase-14 |
|
|
119 |
Matrix Metalloproteinase-16 |
|
|
101 |
Scleraxis ( |
|
|
237 |
Plasminogen Activator Inhibitor 1 |
|
|
116 |
Tissue Inhibitor of Metalloproteinase 2 |
|
|
142 |
Triplicate measurements of transcripts from each cDNA sample were performed using the PerfeCTa SYBR Green FastMix (Quanta Biosciences) according to the manufacturer’s protocol. Briefly, 5 µL of cDNA diluted to 1 ng/µL was combined with 15 µL of a 1∶2 mixture of primers (1.2 µM) and SYBR Green FastMix. The expression of target genes was measured using either the Rotor-Gene Q (Qiagen) or Rotor Gene 6000 (Corbett Research) RT-PCR systems.
The expression of target genes was normalized to the housekeeping gene,
Here,
After calculating the reaction efficiency of each gene, Ct values from the 6% threshold were used to quantify gene expression for the samples according to the following equation, the derivation and validation of which has been described previously
Where
In addition to examining the response of individual genes to TGF-β1, we also developed a novel mathematical analysis for comparing transcript levels between genes that takes into account the primer-specific differences in amplicon sizes. The analysis is based on the assumption that the fluorescence detected by the RT-PCR machine is proportional to the amount of double-stranded DNA (dsDNA) present after each cycle. Primers that code for longer amplicons would therefore have more dsDNA at any given cycle than primers coding for shorter amplicons, and this varies proportionally with amplicon length according to the following relation:
Where
As mentioned previously, we used fluorescence thresholds for each gene that were 6% of the maximum relative fluorescence after 40 cycles. Since all genes were assigned the same relative fluorescence threshold, we set the fluorescence of gene x (
Rearranging and solving for the ratio of the original number of transcripts of genes x and y, we obtain the following equation:
This equation allows us to compare the transcript levels of different genes within individual samples. We can validate this equation by applying it to the traditional comparison of a sample and control group expressing a single target gene (i.e. amplicon size and efficiency are the same). By doing so, we obtain the following:
The right-hand side of the equation is identical to the numerator of
Using
Differences between treatment groups at each time point were determined for area contraction and RT-PCR data using a two-way ANOVA with Bonferroni post tests in Prism (GraphPad Software). In all cases, a p-value of <0.05 was considered to be significant.