Epidemiologic studies have recognized a significantly higher rate of anterior cruciate ligament injury in female athletes than in their male counterparts in various sports. 12,30,50,54 Although the etiology of this phenomena remains unclear, possible explanations include gender differences in ligament or muscle strength, conditioning and endurance, anatomy, and training techniques. 7,25,38,42 Unique to the female athlete is her exposure to constantly changing hormonal milieu throughout her reproductive years. For most of her life, the female athlete is exposed to a rhythmic variation in either endogenous hormones during a regular menstrual cycle or exogenous hormones via oral contraceptives. The cyclic hormonal control mechanisms that regulate the menstrual cycle are driven by estrogen and progesterone. Fluctuations of these sex steroids may in turn translate to changes in metabolism of cells in the anterior cruciate ligament. Realizing that estrogen and progesterone can have individual or interactive effects or both on various metabolic processes, it is possible that estrogen and progesterone also may have an influence on ligament structure, composition, and integrity. 23,44,52
The identification of estrogen and progesterone receptor positive fibroblasts in the human anterior cruciate ligament strongly suggests female sex hormones may have an effect on the structure and composition of the ligament. 22 In a previous study, a dose dependent decrease in cellular proliferation and total collagen synthesis was seen when rabbit anterior cruciate ligament fibroblasts were treated with estradiol for 2 weeks. 23 In a followup study, a similar dose dependent decrease in cellular proliferation and Type 1 procollagen synthesis was seen in female human anterior cruciate ligament fibroblast in the early times (Days 1 and 3) of exposure to increasing levels of estrogen. 53
Like estrogen, progesterone undergoes significant fluctuations during the menstrual cycle. 39 Several studies have reported the complex interaction between estrogen and pro gesterone in relation to connective tissue makeup. 18,32–35,41,52 The effects of constantly changing levels of estrogen and progesterone on ligamentous tissue remain poorly defined. The purpose of this study was to characterize the effects of varying levels of estrogen and progesterone on human anterior cruciate ligament fibroblast proliferation and procollagen synthesis within an in vitro system.
MATERIALS AND METHODS
To compare the effects of physiologic and supraphysiologic levels of exogenously applied estrogen and progesterone on human anterior cruciate ligament fibroblasts, a primary cell culture was established using a modification of the explant culture technique by Nagineni et al. 29 With exposure to logarithmic concentrations of estrogen and progesterone, fibroblast proliferation and rate of collagen synthesis were monitored concurrently using 3H-thymidine incorporation and Types I and III procollagen specific equilibrium radioimmunoassays.
The cell line was obtained from two patients. The first specimen was obtained from a 19-year-old woman undergoing anterior cruciate ligament reconstruction secondary to partial ligament rupture. The patient experienced a twisting injury to her knee. During diagnostic arthroscopy, an anatomically intact but nonfunctional anterior cruciate ligament (stretched) was harvested. The second specimen was obtained from a 32-year-old multiparous woman undergoing total knee replacement secondary to degenerative arthritis. This investigation was approved by the institutional human subjects protection committee, and informed consent was obtained before enrollment.
Western Blot Analysis
To test the efficacy and reliability of the procollagen monoclonal antibodies within a tissue culture system, Western blot analysis was performed. Logarithmic dilutions of certified fetal bovine serum and cell culture homogenates were compared with the radioimmunoassay standards for Types I and Type III procollagens. Specifically, media and cell lysates samples from Day 3 that had received 0.025 ng/mL of estradiol and 1 ng/mL progesterone were homogenized and centrifuged for 5 minutes at 15,000 g. Thereafter, the radioimmunoassay standards, logarithmic dilutions of certified 10% fetal bovine serum (manufacturer specifications indicate 21 pg/mL of estradiol and 50 pg/mL of progesterone in stock fetal bovine serum; Gibco Technologies Inc, Grand Island, NY), and the cell culture supernatant were run in a 10% gradient polyacrylimide gel for 2 hours. After transfer to nitrocellulose paper and blocking of nitrocellulose with nonfat milk, the primary antibody followed by the secondary immunofluorescent tagged antibody were applied separately for 1 hour each at room temperature. The relative banding patterns and intensities for Type I procollagen and Type III procollagen were compared.
Cell Culture Protocol
The anterior cruciate ligament of the subjects was obtained under aseptic conditions in the operating room. Thereafter, the tissue was placed immediately into sterile tissue culture media. All additional manipulations were conducted under a sterile biologic hood. The synovial sheath was excised, and the remaining specimens from both patients were minced finely by transverse sections with a surgical blade and equally divided into two 75-cm2 tissue culture flasks (Corning Glass Works Inc, Corning, NY) containing 15 mL growth medium, which consisted of Minimal Essential Medium-alpha supplemented with certified 10% fetal bovine serum, gentamicin (5 μg/mL), and streptomycin (100 μg/mL).
Primary cell cultures were maintained in humidified atmosphere of 5% CO2 and 95% air at 37° C without a change in media until 1 week after harvest. Thereafter, culture media was changed every 3 days. Cell growth was monitored with an inverted phase contrast microscope. Once cells grew to 90% confluence, the explants were detached with 0.25% trypsin in calcium and magnesium free phosphate buffered saline (pH = 7.4) with 0.1 mmol/L ethylenediaminetetraacetic acid (Gibco Technologies Inc) for 5 minutes at 37° C. At this and subsequent subculturing (P1–P3), the cells were split at a 1:5 ratio.
Estrogen and Progesterone Administration
Fibroblasts cultured in the third passage to confluence were released with trypsin, washed in growth medium supplemented with 10% certified fetal bovine serum, and cultured in 12-well Corning cluster plates at a density of approximately 2 × 104 cells per well. To use accurate experimental techniques, Groups I and II were raised in phosphate buffered solution and 10% fetal bovine serum, respectively, to serve as controls. In an effort to simulate the physiologic and supraphysiologic levels of estradiol and progesterone present in the human menstrual cycle (0.025–0.3 ng/mL for estrogen and 1–11 ng/mL for progesterone) 39 experimental Groups III through XI were supplemented with 10% certified fetal bovine serum and received logarithmic concentrations of 17β-estradiol for 1, 3, 5, and 7 days, as outlined in Table 1. The media with the appropriate concentrations of estrogen or progesterone or both were replaced every 3 days.
Deoxyribonucleic acid (DNA) content and DNA synthesis of control and experimental groups were monitored using 3H-thymidine incorporation of cultures. 29 Confluent cultures at Passage 3, which had been exposed to the respective concentrations of estradiol for 1, 3, 5, and 7 days, were incubated with 10 μL 3H-thymidine (1 μC/mL, New England Nuclear, Boston, MA) for 24 hours at 37° C in a humidity chamber at 5% CO2 and 95% air. Subsequently, agarose was precipitated, and the free 3H-thymidine was removed. The remaining contents were transferred into glass test tubes containing 3 mL of 10% trichloroacetic acid. The contents of the tubes were vortexed and left on ice for 30 minutes to precipitate any macromolecules. Trichloroacetic acid perceptible radioactivity was collected by filtration through Whatman glass microfiber filters using a millipore filtration unit. Filters were air dried, placed into scintillation vials containing 10 mL of scintillation fluid (Liquiscint, National Diagnostics Inc, Manville, NJ) with 10% acetic acid, and the radioactivity was measured in a scintillation counter (Beckman 1801 Beta Counter, Beckman Inc, Pasadena, CA). Cell numbers in replicate of six wells were determined, and the thymidine incorporation was expressed in counts per minute/103 cells.
Quantification of Collagen Synthesis
The effects of varying concentrations of estradiol on procollagen levels, as an indicator of collagen synthesis, were determined using commercially available equilibrium radioimmunoassays. Levels of procollagen Type I were measured with the use of a monoclonal antibody directed against the trimeric carboxy terminal of procollagen I (PICP, Farmos Diagnostica, Turku, Finland), 28 and procollagen Type III levels were measured by a monoclonal antibody specific for the amino terminal of procollagen Type III (PIIINP, Farmos Diagnostica). 40
Confluent cultures at Passage 3, which had been exposed to the respective concentrations of estradiol for 1, 3, 5, and 7 days, were incubated with the respective 125I tracer and antibody for 2 hours at 37° C. Thereafter, the precipitating secondary antibody complex was added to separate the bound tracer from the free tracer element. The tube was centrifuged and decanted after a 30-minute incubation at room temperature. The bound tracer in the pellet was placed into scintillation vials containing 10 mL of scintillation fluid (Liquiscint with 10% acetic acid), and the gamma counts were measured in a scintillation counter (Micromedic Systems 4/600 Automatic Gamma Counter, Micromedic Systems Inc, Horsham, PA). The resulting counts are inversely proportional to the amount of procollagen present in each sample. Results were converted to nanograms per milliliter from the calibration curve derived from the radioimmunoassay standards. Cell numbers in replicate were determined and procollagen synthesis expressed as nanograms per milliliter per 103 cells.
Radiolabeled substrate incorporation is expressed as the mean ± standard error mean. Statistical analysis of cellular proliferation, 3H-thymidine incorporation, and levels of procollagen Types I and III were performed by linear regression analysis and matched sign rank test. A significant level of 0.05 was selected and presented in the form of p values.
Western Blot Analysis
In a previous endeavor, Western blot analysis of Type I and Type III procollagen radioimmunoassays showed numerous significant preliminary results that establish the scientific foundations of the current study. 53 The increased intensity of the banding pattern of the positive control radioimmunoassay standards for procollagen Types I and III evidenced the efficiency of the kits in recognizing increasing concentrations of the respective procollagens. In addition, as seen by the absence of any banding pattern when fetal calf serum was incubated with the anti-Type I procollagen antibodies, it was concluded that serum contains undetectable levels of procollagen. Type III procollagen had a similar banding pattern. As a result, the quantitative measurements obtained through this assay reflect the actual level of procollagen produced by the human fibroblasts of the tissue culture system. 53
As illustrated in Figures 1 to 3, the effects of estrogen and progesterone on fibroblast proliferation, separately and in combination, were measured as a function of 3H-thymidine incorporation. After 1 day of steroid hormone exposure, there was a statistically significant decrease in fibroblast proliferation (p < 0.01) with increasing concentrations of estradiol (Fig 1). In contrast, after 1 day of steroid hormone exposure, a statistically significant increase in fibroblast proliferation (p = 0.02) was observed with increasing concentrations of progesterone (Fig 2). With time, these distinct trends with separate administration of estradiol and progesterone to anterior cruciate ligament fibroblasts became attenuated.
However, when anterior cruciate ligament fibroblasts were administered various combinations of estradiol and progesterone, per the scheme outlined in Table 1, a clear and significant trend in fibroblast proliferation was observed. On Days 1, 3, and 5, there was an overall dose dependent decrease in the proliferation of anterior cruciate ligament fibroblasts with increasing estradiol concentrations regardless of increasing progesterone concentrations (p < 0.01 and p < 0.01, respectively). However, the dose dependent decrease of fibroblast proliferation with increasing estrogen administration was attenuated with increasing levels of progesterone (Fig 3). In addition, when controlling for estrogen concentration, there was a dose dependent increase in the proliferation of anterior cruciate ligament fibroblasts with increasing progesterone concentrations. It was apparent the effects of progesterone are more significant at lower levels of estrogen concentration.
The effects of estrogen and progesterone became less apparent with time. By Day 7, no statistically significant correlation was found between estrogen or progesterone concentrations and fibroblast proliferation (p = 0.37).
Procollagen Type I Synthesis
The effects of estrogen and progesterone on Type I procollagen synthesis are illustrated in Figures 4 to 6. On Days 1, 3, and 5, procollagen synthesis decreased in a dose dependent manner with increasing estrogen concentration regardless of progesterone concentration (p < 0.01). However, the effects of estrogen were attenuated by increasing levels of progesterone. Controlling for estrogen concentration, there was a dose dependent increase in procollagen Type I synthesis on Days 1, 3, and 5. The effects of progesterone on Type I procollagen synthesis were more pronounced at the lower concentrations of estrogen.
Similar to the trends observed in fibroblast proliferation, the effects of estrogen and progesterone on Type I procollagen synthesis became less apparent with time. By Day 7, no statistically significant correlation was found between estrogen or progesterone concentration and procollagen Type I synthesis (p = 0.24 and p = 0.31, respectively).
Procollagen Type III Synthesis
Effects of estrogen and progesterone on Type III procollagen synthesis are illustrated in Figures 7 to 9. No statistically significant differences or general trends in Type III procollagen synthesis were seen with varying estrogen or progesterone concentrations at any time (p = 0.38 and p = 0.41, respectively).
Collagen is produced by fibroblasts and organized in a hierarchical manner from fascicles to microfibrils, whereby it subsequently performs the major load bearing function of the anterior cruciate ligament. 13,17,45 Alterations in the metabolism of fibroblasts, by local stimuli with growth factors 21 or systemic hormonal stimuli, 16 have an influence on the quantity, type, and stability of the collagen in the anterior cruciate ligament. 4,24,53
Collagen is synthesized as a larger molecule with propeptide extensions at both ends that are cleaved stoichiometrically during fibrinogenesis and released into the extracellular fluid. 17,24 The sequence removed from the carboxy terminal end of the molecule, known as the carboxy terminal propeptide, can be found in the extracellular matrix. 28,40 As a result, the extracellular levels of these cleaved extensions, which have been named procollagens by the manufacturer of the equilibrium radioimmunoassays, directly reflect the extracellular levels of collagen. 5 This stoichiometric proportionality has been supported by studies in which levels of Type I and Type III procollagens were increased when collagen synthesis was stimulated by growth hormone therapy. 4,47,49 Similarly, studies of bone biopsy specimens from patients with various forms of metabolic bone disease 36 and in women who are postmenopausal 15,46 have reported that serum Type I procollagen levels correlate significantly with histomorphometric measure of bone formation and density.
It is known that female sex hormones have a widespread effect on the growth and development of bone, muscle, and connective tissues. 9,20,26,44,52 Although numerous animal studies have shown the effect of female sex hormones on collagen metabolism in various tissues, ligaments have not been the subject of much investigation. 3,7,10,11,13,43,44,52 Several in vivo studies have shown that the quantity of collagen in certain tissues is influenced by estrogen. Long-term (in terms of weeks) estrogen administration causes a decrease in the total amount of collagen in the rat hip capsule, skin, aorta, and tail tendon. 11,13,44
Some investigators have shown how local estrogen concentrations may influence connective tissue metabolism. It has been reported that oral estrogen and progesterone therapy significantly reduced Type I procollagen in women who are postmenopausal. 15 Within a similar group of patients, there is an identical increase in total body Type III collagen content with daily administration of estrogen. 14 Estrogen administration is known to acutely decrease total collagen in rat tendon and fascia and to acutely decrease collagen synthesis in rat periodontal tissue. 8,10 In the rat uterus and primate sex skin, estrogen acutely increases newly synthesized collagen, mostly Types I and III, while increasing collagen degradation, with the net result being a dynamic flux between the synthesis and the degradation of collagens. 3,8 Although estrogen administration reduces stiffness in periarticular connective tissue of rabbit knees by approximately 50% compared with untreated animals, 1 it has been shown to decrease hexosamine and soluble collagen content. 52 Thus, it stands to reason that acute fluctuations in the serum estrogen concentration during the menstrual cycle may induce changes in the metabolism and the amount and type of the collagen in the anterior cruciate ligament.
Previous work done in the authors’ laboratory has established the presence of estrogen and progesterone receptors in the human anterior cruciate ligament. 22 The identification of estrogen receptor positive fibroblasts in the human anterior cruciate ligament strongly suggests female sex hormones may have an effect on the structure and composition of the ligament. In one study, a dose dependent decrease in cellular proliferation and total collagen synthesis was seen when rabbit anterior cruciate ligament fibroblasts were treated for 2 weeks with increasing estradiol concentrations. 23 In a followup study, a similar dose dependent decrease in cellular proliferation and Type I procollagen synthesis was seen in female human anterior cruciate ligament fibroblasts in the early times (Days 1 and 3) of exposure with increasing levels of estrogen. Similar to the findings of the current study, no trend in Type III procollagen synthesis was observed with varying levels of estrogen.
Estrogen and its fluctuations in concentration between 0.06 ng/mL and 0.3 ng/mL during the human menstrual cycle may exert an inhibitory dose dependent effect on anterior cruciate ligament fibroblast proliferation and procollagen Type I synthesis. Progesterone is the other major hormone involved with the menstrual cycle. It also undergoes major fluctuations during the menstrual cycle, ranging from 0.95 ng/mL during the follicular phase to 11 ng/mL during the luteal phase. 39 Several studies have described the complex interaction between estrogen and progesterone in relation to connective tissue makeup. 18,32–35,41,52
Yamamuro et al 52 observed a significant reduction in collagen of ovariectomized rats given estrogen with or without progesterone, whereas progesterone alone considerably increased the collagen content. This suggests that estrogen and progesterone alone may have opposing effects regarding collagen synthesis in connective tissue. In combination, although the inhibitory effects of estrogen in fibroblast proliferation and Type I procollagen synthesis appear to predominate, progesterone appears to mitigate its inhibitory effects.
Okulicz and colleagues 32–35 have shown that progesterone downregulates the estrogen receptor in the rodent uterus and primate endometrial stromal cells, the primary connective tissue cell within the uterus. The downregulation of the estrogen receptor is considered an important mechanism whereby estrogen action is inhibited, redirected, or reduced. In addition, different cells within the uterus respond differently to the same hormonal stimulus. Several investigators have observed that endometrial stromal cells are more sensitive to downregulation of the estrogen receptor by progesterone than are glandular epithelial cells in the primate uterus. 27,32–35,37
Samuel et al 41 studied the effects of relaxin on the nonpregnant rat pubic symphysis in conjunction with estrogen and progesterone. Estrogen primed rats produced a greater reduction in total collagen content but had no significant effect on collagen solubility or composition when treated with relaxin. This enhanced effect from estrogen priming was lost when progesterone was added. They concluded that relaxin has potent inhibitory effects on the amount of collagen that is potentiated by estrogen and antagonized by progesterone.
Several end points may exist for the progesterone antagonism of estrogen action. In an attempt to elucidate the molecular mechanism underlying the antagonism, Kraus et al 18 used a model system to analyze the cross talk between the estrogen and progesterone signaling systems. They showed that liganded progesterone receptor complex repress estrogen receptor activity by interfering with its ability to interact productively with the transcriptional machinery through a process known as quenching. As a result, Kraus et al suggested that one of the levels of interplay between estrogen and progesterone actions begins at the basic level of gene expression.
In the current study, accounting for the varying levels of progesterone, a dose dependent decrease in anterior cruciate ligament fibroblast proliferation and procollagen Type I synthesis was observed with increasing levels of estrogen. It appears estrogen has a dominant inhibitory effect on anterior cruciate ligament fibroblast proliferation and procollagen Type I synthesis. These data are consistent with observations made previously in the authors’ laboratory and reported in the literature. The inhibitory effects of estrogen are clearly mitigated by progesterone. Specifically, a dose dependent increase in anterior cruciate ligament fibroblast proliferation and procollagen Type I synthesis was observed when progesterone concentrations were increased as estrogen concentrations were held constant. At lower concentrations of estrogen concentrations (physiologic), the dose dependent effects of progesterone were more pronounced than at the higher estrogen concentrations (supraphysiologic). This phenomenon again confirmed estrogen exerting the more dominant effect.
The effects of estrogen and progesterone were most pronounced in the early periods of hormone exposure—Days 1, 3, and 5. By Day 7, the dose dependent effects of estrogen and progesterone on fibroblast proliferation and Type I procollagen levels became attenuated. Again, this phenomenon with time may be attributable to normal cellular physiology of receptor saturation, cell contact inhibition, or more commonly, downregulation of the receptor protein.
In contrast to Type I procollagen synthesis, no significant changes were observed in Type III procollagen levels with varying doses of estrogen or progesterone with time. These results illustrate that net Type III procollagen synthesis remained minimally affected with estrogen and progesterone exposure, suggesting that presumed differences in fibroblast metabolism are manifested largely by changes in Type I procollagen synthesis, rather than Type III.
The proper function of a ligament depends on the appropriate type, synthesis, assembly, crosslinking, and remodeling of collagen. 6,17,24 This complex interplay between synthesis and remodeling of collagen is influenced by hormones, exercise, and immobilization. 20,31,39,45,48,51 Thus, it is clear the amount of collagen bundles and the individual types of collagen influence the ability of the tendon to withstand loading. It also is known that Type I collagen imparts a great mechanical strength to connective tissues, whereas Type III collagen has been correlated directly with tissue elasticity. 17,24 Although the relative strength of Type I collagen to Type III collagen remains unknown, it generally is agreed that a larger ratio of Type I to Type III collagen in the ligament is indicative of greater strength, whereas a lower ratio may be characteristic of tissue laxity. 19 In applying this understanding to the outlined results, it intuitively follows that the relative decrease in Type I procollagen synthesis with increasing estradiol concentrations could lead to ligament weakening.
The observed causal relationship between changes in relative estrogen and progesterone concentrations and fibroblast proliferation and procollagen synthesis continue to complement the clinical significance of these findings when one considers that female athletes are exposed to rhythmic variations in either endogenous hormones during a menstrual cycle or exogenous, nonphysiologic levels of hormones via the use of oral contraceptives. Although caution should be exercised in extrapolating experimental findings to the clinical situation, taken together these fluctuations in the serum estrogen and progesterone levels translate to acute changes in fibroblast metabolism. The net effect of these cellular events could result in reduced strength of the ligament and subsequently lead to a higher risk of anterior cruciate ligament injury in female athletes.
This study examined the effects of varying doses of estrogen and progesterone on fibroblast metabolism in cell cultures of the human anterior cruciate ligament. The results show that physiologic and supraphysiologic levels of estrogen have a significant dose dependent effect on the fibroblasts of the anterior cruciate ligament. Fibroblast proliferation and Type I procollagen synthesis are reduced significantly with increasing estrogen concentration. However, these effects are mitigated by increasing doses of progesterone. Fibroblast proliferation and Type I procollagen synthesis are increased significantly with increasing progesterone concentration when estrogen levels are held constant. Thus, cumulative or acute fluctuations in serum estrogen and progesterone concentrations or both, such as those occurring during the menstrual cycle or via the additive effect of exogenous estrogen in oral contraceptives, seem to induce changes in the metabolism of anterior cruciate ligament fibroblasts. These structural and compositional changes could result in reduced strength of the anterior cruciate ligament and predispose athletic females to ligament injury.
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