Combined effects of estrogen and progesterone on the anterior cruciate ligament.

Previous studies from the authors' laboratory have established the presence of estrogen and progesterone receptors in the human anterior cruciate ligament. The purpose of the current study was to investigate the combined effects of 1beta-estradiol and progesterone on cell proliferation and procollagen synthesis of the human anterior cruciate ligament fibroblasts. Fibroblast proliferation and procollagen synthesis in response to logarithmic concentrations of 17beta-estradiol (0.0025 ng/mL, 0.025 ng/mL, 0.25 ng/mL) and progesterone (1 ng/mL, 10 ng/mL, 100 ng/mL) were assessed with the measurement of 3H-thymidine incorporation and Types I and III procollagen specific equilibrium radioimmunoassays. On Days 1, 3, and 5 there was a dose dependent decrease in the fibroblast proliferation and procollagen Type I synthesis with increasing estradiol concentrations. The effect was attenuated with increasing progesterone concentrations. Controlling for estrogen levels, a dose dependent increase in fibroblast proliferation and procollagen Type I synthesis was observed with increasing progesterone concentrations. The effect was more pronounced at lower concentrations of estrogen, suggesting estrogen levels were the dominant factor. The effects of estrogen and progesterone became less apparent by Day 7. No significant differences in Type III procollagen synthesis were seen with varying estradiol concentrations at any of the designated times. These early physiologic changes in fibroblast proliferation and Type I procollagen synthesis may provide a biologic explanation for the increased anterior cruciate ligament injury rate observed in female athletes, suggesting the acute cyclical hormonal variations in the female athlete during menstruation predispose her to ligamentous injury.

Effect of estrogen on cellular metabolism of the human anterior cruciate ligament.

Previous studies from this laboratory have established the presence of estrogen receptors in the human anterior cruciate ligament. The purpose of this study was to investigate the effects of 17 beta-estradiol on cell proliferation and procollagen levels, as an indicator of collagen synthesis, in the human anterior cruciate ligament fibroblasts. Fibroblast proliferation and procollagen synthesis in response to near log concentrations of 17 beta-estradiol (at 0.0029 ng/mL, 0.025 ng/mL, 0.25 ng/mL, 2.5 ng/mL, and 25 ng/mL) were assessed with the measurement of 3H-thymidine incorporation and Types 1 and 3 procollagen specific equilibrium radioimmunoassays. On Days 1 and 3, there was a dose dependent decrease in the proliferation of anterior cruciate ligament fibroblasts with increasing estradiol concentrations. This dose dependent effect of decreased fibroblast proliferation with increasing estradiol concentrations became less apparent at 7, 10, and 14 days. On Days 1 and 3, procollagen synthesis decreased in a dose dependent manner with increasing estradiol concentrations. On Days 7, 10, and 14, this dose dependent effect was attenuated. No significant differences in Type 3 procollagen synthesis by anterior cruciate ligament fibroblasts were observed with varying estradiol concentrations at any of the designated points. These early physiologic changes in fibroblast proliferation and Type I procollagen synthesis may provide a biologic explanation for the increased anterior cruciate ligament injury rate observed in female athletes, suggesting that it is the acute cyclic variations in the female athlete who is menstruating that predisposes her to ligamentous injury.

Fibroblast growth factor and epidermal growth factor receptors in ligament healing.

Although recent in vivo studies indicate that basic fibroblast growth factor hastens the healing and strength of the medical collateral ligament after injury, in vitro studies with epidermal growth factor and basic fibroblast growth factor have shown increased fibroblast proliferation with the exogenous administration of these growth factors. Using an established spontaneously healing rabbit injury model, the surgical transection of the medial collateral ligament was undertaken in 12 anesthetized male adult rabbits. Immunohistochemical localization using monoclonal antibodies to the basic fibroblast growth factor receptor and epidermal growth factor receptor were used to identify the distribution and relative concentrations of the individual receptors at 3, 7, 14, and 28 days after surgery. Realizing that the trophic effects of basic fibroblast growth factor and epidermal growth factor are determined exclusively by their individual receptors, this study confirms the hypothesis that basic fibroblast growth factor and epidermal growth factor receptor proteins are present and increase during the early stages of medial collateral ligament healing. The understanding of this rabbit ligament injury model has far reaching implications to ligament healing seen in humans. By elucidating the spatial and temporal regulation of the basic fibroblast growth factor and epidermal growth factor receptor proteins, exogenous growth factor therapy, once approved for human use, potentially can be synchronized with maximal protein receptor levels.

Estrogen affects the cellular metabolism of the anterior cruciate ligament. A potential explanation for female athletic injury.

Investigations from this laboratory have established the presence of estrogen receptors in the human anterior cruciate ligament. This study further investigates the effects of 17 beta-estradiol on the cellular proliferation and collagen synthesis of fibroblasts derived from the rabbit anterior cruciate ligament. Fibroblast proliferation and collagen synthesis in response to near log concentrations of 17 beta-estradiol (at 0.0029, 0.025, 0.25, 2.5, and 25 ng/ml) were assessed by measuring [3H]thymidine and [14C]hydroxyproline incorporation, respectively. Collagen synthesis was significantly reduced with increasing local estradiol concentration (P < 0.001). Declining collagen synthesis was first noted at a 17 beta-estradiol concentration of 0.025 ng/ml. Within normal physiologic levels of estrogen (0.025 to 0.25 ng/ml), collagen synthesis was reduced by more than 40% of control, and at pharmacologic levels of 2.5 and 25 ng/ml, by more than 50% of control. A significant reduction of fibroblast proliferation was also observed with increasing estradiol concentrations (P = 0.023). Clinically, alterations in anterior cruciate ligament cellular metabolism caused by estrogen fluctuations may change the composition of the ligament, rendering it more susceptible to injury.

Morphology and matrix composition during early tendon to bone healing.

This study outlines the early morphologic phenomenon of tendon to bone healing in the rabbit model. Twelve skeletally mature, male New Zealand White rabbits received transplantation of the hallucis longus tendon into a 2-mm calcaneal bone tunnel. The morphologic characteristics of the healing tendon to bone interface were evaluated at 1, 2, 4, and 6 weeks after surgery by the use of conventional histology and immunohistochemical localization of collagen Types I, II, and III. Histologic analysis illustrated progressive maturation and reorganization of the tendon to bone interface with subsequent development of tissue collagen fiber continuity between the tendon and bone. Initially, diffuse immunolocalization of all three collagen types was observed within the scar tissue filling the space between the tendon and bone. During a 6-week period, reorganization of the scar tissue into an interface occurred, similar to an indirect insertion. Although a definitive fibrocartilage region did not form, Type II collagen was localized at the remodeling insertion site throughout the first 6 weeks of repair. In addition, Type III collagen fibers, resembling Sharpey's fibers, were noted to span this interface. The characterization of the insertion between tendon and bone is important to the understanding of healing in commonly used orthopaedic grafting procedures, such as anterior cruciate ligament reconstructions.

Primary immunolocalization of androgen target cells in the human anterior cruciate ligament.

To identify androgen target cells in the human anterior cruciate ligament, immunohistochemical localization of the androgen receptor was performed in 31 specimens of the ligament. All of the specimens were obtained at surgery. Seventeen specimens were from women, and 14 were from men: the average age of the patients was 45.2 years (range: 18-78 years). An immunoperoxidase method using monoclonal antibodies to the androgen receptor was employed to identify androgen target cells in the ligament. Consistent staining of the androgen receptor was demonstrable in six specimens obtained from young men 18-24 years old, and equivocal positive staining was seen in two other specimens from young men. No receptors were demonstrated by this method in any of the specimens from women or older men. Androgen receptors were localized to synoviocytes in the synovium and under the synovial lining, fibroblasts in the ligament stroma, and cells lining the blood-vessel walls of the anterior cruciate ligament. The demonstration of androgen receptors in the cells of the anterior cruciate ligament strongly suggests that male sex hormones may have an effect on the structure and composition of this ligament in young men.

Primary immunolocalization of estrogen and progesterone target cells in the human anterior cruciate ligament.

To identify estrogen and progesterone target cells in the human anterior cruciate ligament immunohistochemical localization of both estrogen and progesterone receptors was performed in 17 specimens of human anterior cruciate ligament. All ligament specimens were obtained at surgery. Thirteen specimens were from women, and four were from men: the average age was 57 years (range, 18-78 years). Eleven specimens (from nine women and two men) came from total knee replacements for osteoarthritis of the knee: three (from two women and one man), from reconstructions of the anterior cruciate ligament: two (both from women), from medial meniscectomies; and one (from a man), from an amputation secondary to chondrosarcoma of the pelvis. An immunoperoxidase method using monoclonal antibodies to the estrogen and progesterone receptors was employed to identify estrogen and progesterone target cells in the anterior cruciate ligament. Staining of both receptors was demonstrable in 14 specimens and in the remaining three specimens less than 15% of the cells were stained. Both estrogen and progesterone receptors were localized to synoviocytes in the synovial lining, fibroblasts in the anterior cruciate ligament stroma and cells in the blood vessel walls of the ligament. This demonstration of receptors for estrogen and progesterone in the cells of anterior cruciate ligament suggests that female sex hormones may have an effect on its structure and composition.

Testosterone-induced copulatory behavior is affected by the postcastration interval.

A well entrenched hypothesis regarding hormonal action is that as the time interval following hormonal deprivation increases there is a corresponding decrease in the sensitivity of the system to the effects of hormone replacement. With this in mind, we examined the effects of a prolonged period of hormonal deprivation (9 mo), and compared these to the effects of a shorter period (1 month), on the restoration of copulatory behavior and seminal vesicle weights. Castration of sexually vigorous male Long-Evans rats at 6 mo of age was followed by the virtual disappearance of ejaculatory behavior within 1 mo. Testosterone (T) was administered (5 mm or 20 mm T-containing Silastic capsules) either 1 or 9 mo after castration, and copulatory tests were conducted 3, 7, 10, 14, and 17 days later. 5 mmT and 20 mmT were equally effective in restoring behavior in the rats treated 1 mo after castration. In contrast, 5 mmT was more effective in inducing copulatory behavior than 20 mmT in the rats treated 9 mo after castration. The time course to maximal effect was longer in the rats given T 9 mo after castration. Rats were sacrificed 21 days after T administration. Expressed seminal vesicle weights and plasma testosterone were increased in a dose-dependent manner independent of the postcastration interval. These data indicate that somatic and behavioral effects of T are differentially modified by the period of preceding hormonal deprivation.