Effects of hind limb unloading and reloading on nitric oxide synthase expression and apoptosis of osteocytes and chondrocytes.

In rat bone, the absence of mechanical load results in a reduction in bone formation, inhibition of longitudinal growth, and a decrease in the number of osteoblasts and osteoprogenitors in cancellous bone. Unloading has also been linked to an increase in apoptosis of osteocytes and chondrocytes through production of nitric oxide (NO) and increased expression of NO synthases (NOS). Reloading results in recovery of bone volume within 14 days, although osteoblast and osteoclast numbers remain below control values, suggesting decreased bone turnover. This study was designed to evaluate the effects of hind limb unloading and subsequent reloading on apoptosis, NOS expression, and histomorphometric parameters in trabecular and cortical bone, articular cartilage, and growth plate cartilage of the proximal tibia of the hind limbs. Compared to ambulatory controls, 2 weeks of unloading resulted in a 66% increase in the percentage of apoptotic osteocytes in the trabecular metaphysis, a 14% increase in osteoclast number and a 48% decrease in bone volume. The percentage of eNOS- or iNOS-positive osteocytes was unchanged. Upon reloading, the percentage of apoptotic osteocytes and bone volume returned to baseline whereas the percentage of iNOS-positive osteocytes increased by 50% and osteoclast number decreased by 30% compared to ambulatory controls. More striking changes were observed in articular and growth plate cartilage. Unloading resulted in a 230% increase in apoptotic chondrocytes, a 400% increase in iNOS-positive chondrocytes, and a 17% reduction in width in articular cartilage. Reloading for 2 weeks resulted in partial recovery. Chondrocytes in the proliferative and hypertrophic zones of the growth plate responded similarly to those in the articular cartilage. In summary, we observed that 14 days of unloading increased apoptosis of osteocytes and chondrocytes. This was associated with an increase in the proportion of iNOS-positive chondrocytes whereas the proportion of iNOS-positive osteocytes remained unchanged. Reloading for 14 days restored osteocyte apoptosis to control levels but the percentage of iNOS- and eNOS-positive osteocytes increased in reloaded bone compared to controls. This was associated with a decrease in osteoclast number. In cartilage, reloading for 2 weeks did not result in a return to baseline in any of the parameters measured, suggesting that the effects of unloading on articular cartilage and the growth plate last longer than those in bone and may have prolonged effects on joint biomechanics and longitudinal bone growth.

The effect of reloading on bone volume, osteoblast number, and osteoprogenitor characteristics: studies in hind limb unloaded rats.

Skeletal unloading during space flight results in bone loss. In astronauts the extent to which bone is lost varies greatly between different bones of the skeleton as well as between different individuals. Following return to earth, recovery of bone mass during reloading also varies between different bones and different individuals. Due to this variability and the limited number of subjects it is difficult to study the effects of unloading/reloading on bone in humans. A viable alternative is to use the rat model of hind limb unloading developed at NASA. We have previously demonstrated that, in 6-week-old male rats, 14 days of unloading result in a decrease in osteoprogenitor number in cell populations isolated from the proximal femur. The goal of the present study was to determine the number of osteoprogenitor cells present in cell populations derived from the proximal femur of young rats after 14 days of unloading followed by 14 days of reloading and to characterize their proliferative capacity. To do this, we determined the number of alkaline phosphatase-positive colony forming units (CFU-AP) and osteoblast CFU (CFU-O). To establish whether the effects of unloading and reloading were specific for cells of the osteoblast lineage, we also determined the number of fibroblastic CFU (CFU-F). Effects on proliferation were evaluated by measuring the size of CFU-O. Unloading resulted in a 66% reduction in CFU-AP. CFU-O numbers were decreased by 76% and mean colony size was 33% less than controls. The decrease in osteogenic and osteoprogenitor cells in vitro paralleled the decrease in bone volume (- 50%), osteoblast number (- 35%), and bone formation rate (- 46%) observed in the proximal tibial metaphysis of unloaded rats. Unloading had no effect on osteoclast number or surface. Subsequent reloading for 14 days restored CFU-AP. CFU-O numbers were only partially restored at 14 days (83% of controls) but nodule size was 1.2-fold greater than controls. Neither unloading nor reloading had an effect on the total number of progenitors (CFU-F). Reloading restored bone volume back to control values, but osteoblast number and bone formation rate were still lower than those in corresponding controls. Both osteoclast number and surface were lower in reloaded animals than in age-matched controls. Our results indicate that 14 days of unloading result in a decrease in osteoprogenitor number and that reloading for 14 subsequent days completely restores CFU-AP and bone volume to control levels, while the number of CFU-O in vitro and osteoblasts in vivo were partially recovered but still lower than corresponding controls. Strikingly, osteoclastic bone resorption after 14 days of reloading was greatly reduced compared to controls, suggesting a significant contribution of this to the recovery process.