Session V - Fracture Healing

Estrogen Increases Cartilage Formation and Fracture Healing Strength in OVX Rats

Mark E. Bolander, MD; James T. Bronk; Gobinda Sarkar, PhD, Mayo Clinic, Rochester, MN

Purpose: The protective effect of estrogen on bone mass in post-menopausal women is well documented; however, the effect of estrogen depletion on bone formation and repair after fracture has not been evaluated. We report the effect of OVX with and without estrogen replacement on healing of experimental fractures in the rat femur.

Methods: Fracture healing was studied in 4 groups of mature (350 gm) female Long Evans rats. Closed femoral fractures were made by standard methods (Bonnarens and Einhorn). Ovariectomy was performed by the animal supplier (Harlan-Sprague-Dawly). Animals were divided into 5 groups: 1 group with sham ovariectomy and 4 groups with ovariectomy. After ovariectomy, animals were housed for 3 months prior to fracture to allow stabilization of bone remodeling. Animals were pair fed across all 5 groups to control for the effect of ovariectomy on animal appetite and by implication protein intake. Estrogen was delivered by daily subcutaneous injection in sesame seed oil at concentrations of 1, 25, and 50 mg/kg/day. Sham animals and ovariectomized animals who did not receive estrogen were also given daily injections of sesame seed oil. Animals were sacrificed for evaluation 4 days, 7 days, 10 days, 14 days, 3 weeks, and 5 weeks after fracture. Specimens were obtained for mechanical testing, histology, or RT-PCR analysis of estrogen receptor gene expression. Statistical analysis was performed by ANOVA. When the F-test was positive, a significance level was set at P = 0.05. Estrogen receptor messenger RNA levels in the fracture callus of sham-operated animals were detected and quantified by RT-PCR amplification in the presence of radiolabeled nucleotides. The sequence of the PCR product was verified by restriction enzyme digestion.

Results: We studied in vivo the effects of daily estrogen injections on the mechanical properties of healing fracture callus in adult female rats that were osteoporotic secondary to ovariectomy. Ovariectomy resulted in a significant decrease in the mechanical properties of the fracture callus 3and 5 weeks after fracture (P <0.02). Thirty days of treatment with estrogen (1-50 mg/kg/day begun at the time of fracture) resulted in a significant, dose-dependent increase in fracture callus tensile strength (31%), stiffness (50%), and ultimate strength (35%) compared to fractures from untreated animals after ovariectomy (P < 0.04). Estrogen receptor message was present in fracture callus at levels up to 70% of that found in the rat uterus, and demonstrated a transient 13-fold increase in transcription by day 14 of fracture repair.

Discussion and Conclusion: Demonstration of physiologically significant levels of rat estrogen receptor mRNA expression in callus provides a basis for estrogen to play an important and direct role in modulating fracture repair. Furthermore, in vivo data demonstrating a substantial increase in the mechanical properties of healing callus suggests that short-term estrogen treatment begun at the time of fracture in post-menopausal women who are not already on estrogen replacement therapy may improve the strength and speed of early fracture repair. Such a phenomenon would potentially allow earlier mobilization in a patient group with great morbidity from prolonged immobilization. These findings also have implications for the prevention and treatment of nonunion in post-menopausal patients.