Using an in vitro
model of tendon healing, we examined the effects of TGF-β1 on the expression of extracellular matrix proteins, MMPs, MMP inhibitors, and neotendon genes relevant to tendon development, healing and adhesion formation. Our goal was to better characterize the effects of TGF-β1 on tenocyte gene expression, which to date remain mostly not well understood despite some preliminary reports 
, in order to both identify new targets for the prevention of flexor tendon adhesions as well as facilitate future experimentation to understand the molecular underpinnings of TGF-β1’s fibrotic effects. Interestingly, while tenocytes contracted collagen gels to a similar degree after treatment with 1–100 ng/mL of TGF-β1, each level of treatment elicited measurably different responses on gene transcription. In most instances, however, the effects of 10 ng/mL of TGF-β1 on gene expression were similar to those of 100 ng/mL, albeit with some differences in the magnitude and duration of the effect. Nevertheless, the data suggests that 10 ng/mL TGF-β1 is a sufficient dose to elicit the cellular events which caused an increase in fibronectin, collagen, biglycan, PAI-1, Mohawk, and Scleraxis gene transcription as well as MMP-16 and decorin downregulation similar to that which was observed in the 100 ng/mL TGF-β1 gels. We cannot, on the other hand, say the same about the 1 ng/mL dose, which often more closely resembled the control media at a gene transcription level, especially 24 to 48 hours after treatment.
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 
. Similarly, we did not initially expect TGF-β1 to increase the expression of both Scleraxis and Mohawk, transcription factors that play a crucial role in tendon development. Instead, we found that TGF-β1 elicited significant, 6- to 12-fold increases in their expression over control gels. The finding that Scleraxis is upregulated by TGF-β1 in adult tendon cells is consistent with reports that TGF-β1’s role in tissue fibrosis is associated with increased Scleraxis expression in heart 
and muscle 
. Therefore, the hypothesis that increased Scleraxis expression is beneficial to tendon healing warrants further investigation. In any case, the finding that TGF-β1 increases Collagen V, XII, Scleraxis and Mohawk expression indicate that TGF-β1 exerts important regenerative effects on tendon healing that must not be neglected. This is consistent with in vivo
animal models where disrupted TGF-β1 signaling resulted in weaker tendon repairs 
, while over-expression of TGF-β1 was reported to have improved the strength of repairs in a rabbit Achilles tendon healing model 
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 
). As TGF-β1 is associated with scar tissue formation, we hypothesized that it would inhibit or have little effect on proteoglycan expression. However, TGF-β1 stimulated upregulation of biglycan and downregulation of decorin expression in a dose-dependent manner. TGF-β1 also stimulated a moderate upregulation of lumican expression at 1 and 10 ng/mL, but not at 100 ng/mL. As biglycan is present early in tendon development, but eventually gets replaced by increasing amounts of decorin 
, the balance of proteoglycan expression in healing tendon may be a therapeutic target that warrants further investigation. These findings, taken together with reports that biglycan upregulation is associated with tendinopathy 
, suggest that TGF-β1 may also play an important, but currently undefined role in other conditions that are linked with inflammation 
. Finally, the inter-gene analysis of proteoglycan expression revealed that biglycan was expressed much more highly than decorin and lumican in the tenocyte-seeded collagen gels. This finding agrees with developmental data that biglycan is most abundant in the early stages of collagen organization, while decorin increases as fibrils mature 
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 
. Despite the time-dependent upregulation of MMP gene expression, the inter-gene analysis of ECM and MMP expression revealed that 10 and 100 ng/mL of TGF-β1 clearly increased the ratio of ECM gene expression to MMP gene expression. In addition, the upregulation of the MMP activity inhibitor, PAI-1, by all three doses of TGF-β1 as early as 6 hours post treatment is consistent with prior reports implicating PAI-1 modulation of MMP-2 activity, rather than MMP-2 transcriptional downregulation, in TGF-β1 mediated renal fibrosis 
and provides support to our hypothesis that TGF-β1 causes increased ECM production and decreased ECM turnover, leading to the accumulation and persistence of adhesions. These findings warrant formal investigation of the effects of TGF-β1 on MMP activity in future studies.
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 
. This finding suggests that increasing MMP-16 expression may help modulate TGF-β1 mediated healing by directing proper regeneration of the tendon microstructure and/or promoting the degradation of undesirable ECM components, which remains to be formally validated.
Finally, the inter-gene analysis of ECM genes strikingly revealed that the tenocytes seeded in collagen I gels produced very high levels of fibronectin (Fn1
) and collagen III (Col3a1
). Given that the vast majority of mature tendon ECM consists of collagen I 
, this appeared to be a counterintuitive observation. However, since fibronectin and collagen III are known to be highly upregulated during tendon healing 
, their high expression in the collagen gel model adds support to the applicability of 3D collagen hydrogels as an in vitro
model of tendon repair. In addition, the inter-gene analysis of the transcription of ECM proteins and MMPs made it clear that higher doses of TGF-β1 tilted the balance of expression in favor of the ECM genes, a possible means by which TGF-β1 contributes to adhesion formation. This novel inter-gene analysis methodology has immense potential to lend valuable insights into countless gene expression experiments.
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 the vast majority of tendon cells used for this study exhibited the elongated, fibroblast-like morphology typical of tenocytes, we have referred to them as such. Another limitation of this study was that the effects of mechanical stress variations within the pinned collagen gels were not evaluated. It is known that mechanical forces accumulate throughout the process of cell-mediated collagen gel contraction 
. In our model, it is likely that tenocytes contracting the gels around the screws experienced compressive forces while tenocytes between the screws were in tension during gel contraction. As both the outer and inner parts of the gel were pooled for RNA analysis, their combined expression was assessed in this study. It is important to note, however, that the geometry of the pinned collagen gels was such that the vast majority of tenocytes were between the screws and therefore experiencing tension.
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 
and non-Smad pathways 
. Therefore, future studies are warranted to examine the posttranscriptional regulation of ECM and MMP-related genes in tenocytes as well as the signaling pathways through which TGF-β1exerts its pro-scarring effects.
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.