Assistant Professor Boston University Boston, Massachusetts, United States
Introduction: : There are well documented sex differences in tendon injury rates [1,2] and healing outcomes [2,3], yet the underlying mechanisms are still unknown. Given the increased prevalence of injuries and disease associated with the onset of both puberty and in the post-menopausal period, sex steroid hormones are suspected to contribute [5]. Estrogen and progesterone receptors are expressed in both male and female tendons, however studies investigating their individual roles in tenocyte health are limited [5,6]. Prior work in our lab demonstrated that the effect of sex hormones on tendon health, biosynthesis, and ECM content is dependent on mechanical environment, hormone studied, and sex [7]. However, it’s not clear whether these differences are due to chemical processing of hormone or downstream effects. We have established a highly sensitive and quantitative technique to quantify cellular uptake of estrogen and progesterone in live tendon explants. The objective of this study is to determine how estrogen and progesterone pretreatment and mechanical loading influence hormone incorporation into tenocytes, with the goal of uncovering upstream factors that may modulate subsequent processing and signaling. Based on existing literature [5,8], we hypothesized that both estrogen and progesterone would exhibit increased incorporation with same hormone pretreatment, and that injury-like stress deprivation would elicit more hormone uptake than physiologic static strain.
Materials and
Methods: : Flexor digitorum longus (FDL) tendons were harvested from young adult (4 month old) female C57BL/6J mice following a seven-day acclimation period to normalize for stress-induced hormonal changes associated with transportation. Explants were cultured in no hormone (NH), 1nM estrogen-pretreated (EST, 17β-estradiol), or 1nM progesterone-pretreated (PRO) medium for two days in stress deprivation or under static strain. Samples under static strain were gripped with a 10 mm gauge distance, tensioned to a preload of 20g, and displaced an additional 300 µm to induce a 3% strain. On the last day of culture, 3H-estradiol (2.1 μCi/mL, 1μM) or 3H-progesterone (1.94 μCi/mL, 1μM) diluted in NH media was added to culture wells for 30 minutes to quantify the incorporation of hormone into the tendon. Explants were divided into 12 groups (n=4) covering all possible combinations of culture medium, radiolabel, and strain state (Figure 1). Wet and dry weights were recorded for each explant before completing an overnight Proteinase K digest. Radiolabeled hormone incorporation was quantified from digests via scintillation and normalized to dry weight. Incorporation data was analyzed for independent effects of strain state, pretreatment, and hormonal cross interactions. Statistical analysis was performed with one-way ANOVAs with post-hoc t-tests where appropriate. Significance is reported at p< 0.05.
Results, Conclusions, and Discussions:: In support of previous literature, we found that hormone incorporation is influenced by both mechanical loading [6] and hormone exposure [5,8]. Static strain decreased estradiol and progesterone incorporation (Figure 2A-B). Interestingly, static strain did not affect progesterone incorporation if there was no pretreatment, (Figure 2B) suggesting the strain-dependency of estradiol incorporation is independent of pretreatment while strain-dependency in progesterone incorporation may be dependent on previous progesterone stimulation. Inhibiting mechanosensing or studying altered strain states may help identify the mechanisms underlying receptor sensitivity to strain, and this is planned in future studies. Estrogen-pretreated samples exhibited a priming-like effect, leading to increased estradiol incorporation under stress deprivation (Figure 2C). In contrast, progesterone pretreatment exhibited a dampening-like effect, decreasing progesterone incorporation but only in static strain conditions (Figure 2D). This suggests differences in hormone signaling mechanisms between homeostasis and injury, which may contribute to the strain-dependent impacts of sex hormones on ECM remodeling observed in prior work [7] It will be prudent in the future to determine uptake mechanisms (through receptor or transmembrane) as a means of clarifying whether receptor availability or levels are contributing to this phenomena. Interestingly, estradiol and progesterone incorporation were both decreased with opposite hormone pretreatment under static strain, exhibiting antagonistic interactions (Figure 2E-F). However, there were no cross reactions of hormones observed in stress deprived samples (Figure 2E-F). This suggests complex, and underexplored, interactions between the two hormones that could explain phase-dependent changes during the menstrual cycle. Future work will clarify whether these effects are due to chemical interactions and direct influence of receptor-ligand binding. Additional phases of the hormone incorporation, processing, and signaling cascade should also be examined to identify other processes specifically influenced by mechanical loading and hormone exposure. Overall, this work confirms that the ability to process sex steroid hormones is modulated by mechanical loading and hormone exposure, offering a framework for investigating the dynamics of hormone signaling and a step towards understanding complex phenomena such as injury variability across the menstrual cycle.
ACKNOWLEDGEMENTS: This work was supported by funding from the NIH NIGMS (R35-GM151127), Wu Tsai Human Performance Alliance, and National Science Foundation Research Traineeship (NRT) in Biological Feedback Control (NSF-DGE #2244366).
