Prolonged and Repeated Upright Posture Promotes Bone Formation in Rat Lumbar Vertebrae

Study Design. Both forelimbs of rats were amputated and these rats were kept in the custom-made cages to keep prolonged and repeated upright posture. Changes of bone were observed in the lumbar vertebrae at three different time points after the surgery. Objective. To investigate the effect of prolonged and repeated upright posture on bone formation of rat lumbar vertebrae. Summary of Background Data. Previous studies show long-term and repeated load-induced bone formation, but there are no clear evidences to indicate whether or not long-term and repeated assumption of the upright posture could result in bone formation at rat lumbar vertebrae. Methods. The forelimbs of 30 rats were amputated when they were 1 month old. These rats were kept in the custom-made cages and were forced to stand upright on their hindlimbs and tails to obtain water and food. Normal rats of the same ages kept in regular cages were used as control. The rats were sacrificed at 5, 7, and 9 months after the surgery and lumbar vertebrae samples were harvested for micro-computed tomographic (CT), histological, and immunohistochemical studies. Total RNA isolated from these samples were used for real-time polymerase chain reaction of type I collagen (Col1 alpha 2), type X collagen (Col10 alpha 1), transforming growth factor-beta 1 (TGF-beta 1) and its related nuclear transcript factor runt-related transcription factor 2 (Runx2), as well as the biomarker of angiogenesis and vascular invasion, which is also a prerequisite for endochondral bone formation: vascular endothelial growth factor (VEGF). Results. Micro-CT and histological studies showed increased trabecular bone density and increased osteoblast quantities of lumbar vertebrae after surgery. Immunostaining revealed increased protein expression of type I collagen, type X collagen, TGF-beta, and VEGF. Real-time polymerase chain reaction showed upregulated expression of Col1 alpha 2, Col10 alpha 1, VEGF, TGF-beta 1, and Runx2 mRNA. Conclusion. Upright posture induces bone acquisition in the rats' lumbar spine, primarily through the mode of the endochondral ossification, which is associated with increased loading, activated VEGF, and TGF-beta 1 signaling.