Experimental model of osteoporosis on 14 weeks old ovariectomised rats: biochemical, histological and biomechanical study

Abstract

Summary. Ovariectomy is one of the most common ways of inducing experimental osteoporosis, since there are no meaningful differences in bone behavior between surgical and natural menopause. Because ovariectomized rat still represent the “gold standard” in studies of postmenopausal osteoporosis, the present study focuses on an animal model of osteoporosis which we developed for researching the effects of medicament and non-medicament treatment of this bone disease. Experiments were conducted on a total of 18 female Wistar rats aged fourteen weeks, randomly divided into three experimental groups: intact (INT) group and two groups of ovariectomized (OVX) animals (OVX I and OVX II), consisting of 6 animals each. Animals are ovariectomized using a ventral approach, which in our experience is more favorable than the midline dorsal skin incision or double dorsolateral approach, both of which are also commonly used for inducing experimental osteoporosis. The OVX I group consisted of bilateral ovariectomized females sacrificed six weeks after ovariectomy, while OVX II was sacrificed eleven weeks after ovariectomy. The group of intact, un-operated control animals (INT group) was sacrificed after eleven weeks together with animals of the OVX II group. Blood samples for measurement of serum alkaline phosphatase, osteocalcin, calcium and phosphorus level are collected from animals of the INT and OVXI group after six weeks and from animals of INT and OVX II groups after eleven weeks. Histological analyses were done on tibia while biomechanical measurements were done on the femurs of animals from INT, OVXI and OVX II groups. In animals of the OVX I group, levels of alkaline phosphatase, osteocalcin, phosphorus and calcium were statistically significantly increased (p < 0.01) compared to intact female (INT group) controls, indicating an increase in bone degradation. Histological analysis shows that the tibia of ovariectomized females are characterized by the appearance of fine thinned trabecular bone, with enlargement of Haversian channels in cortical bone and with gradual transition of lamellar (cortical) bone to cancellous (trabecular) bone. Biomechanical measurements also confirm significant changes in the quality of the bone after bilateral ovariectomy and show increasing fragility of the femur in the biomechanical bending test. In females that were sacrificed eleven weeks after ovariectomy (OVX II group), biochemical parameters also suggest accelerated bone degradation, but signs of stabilization of resorptive activity were present. Levels of alkaline phosphatase, osteocalcin, phosphorus and calcium were significantly increased in OVXII (p < 0.01) vs. INT, but were very close to those measured in the OVX I group. Histological findings for OVXII also indicate loss of longitudinal trabeculae, extension of the medullary canal and extension of the area with bone marrow 11 weeks after bilateral ovariectomy. This is consistent with the bone becoming more porous and increasingly fragile, resulting in lower and lower bone mass, with increasing holes and spaces. In agreement with this, as the bones become more porous, significantly reduced moment of force and deflection in bending was detected. With this experiment we demonstrated that ovariectomy using a ventral approach is a preferable method which provides 100 percent survival of animals as well as rapid wound healing without complications after the surgical procedure, while results obtained by histological and biomechanical analysis of the bones of ovariectomized animals reliably confirmed the existence of osteoporotic processes that progress over time. Thus, our animal osteoporotic model is suitable for testing the effects of drugs and physical procedures in postmenopausal osteoporosis.