Osteoporosis influences the early period of fracture healing in a rat osteoporotic model.

Osteoporotic fractures commonly occur in the elderly. Although current therapies are aimed at the prevention and treatment of osteoporotic fractures, studies examing the fracture healing process in osteoporotic bone are limited. We produced an osteoporotic rat model by ovariectomy (ovx) and maintained a low calcium diet (LCD) in order to evaluate the influence of osteoporosis on fracture healing. Callus formation and strength was monitored over a 3 week period by histological and biomechanical assessment. Data collected simultaneously on a group of rats undergoing sham surgery (sx) were used for comparison. A 40% reduction in fracture callus cross-sectional area and a 23% reduction in bone mineral density in the healing femur of the ovx rats was observed on day 21 following fracture as compared with the sx group (p < 0.01). Biomechanical data from the healing femur of the ovx rats revealed a fivefold decrease in the energy required to break the fracture callus, a threefold decrease in peak failure load, a twofold decrease in stiffness and a threefold decrease in stress as compared with the sx group (p < 0.01, respectively). Histomorphological analysis revealed a delay in fracture callus healing with poor development of mature bone in the ovx rats. This study provides physical evidence of altered fracture healing in osteoporotic bone, which may have important implications in evaluating the effects of new treatments for osteoporosis on fracture healing.

Nitric oxide regulates alkaline phosphatase activity in rat fracture callus explant cultures.

Nitric oxide (NO) is synthesised by a group of enzymes called nitric oxide synthases (NOS) and oxidizes to its stable end-products nitrite (NO2-) and nitrate (NO3-) We have previously reported in an in vivo rat model that NO is an important regulator for rat bone fracture healing. This study examines the effects of NO on alkaline phosphatase (ALP) activity in a rat fracture callus explant culture system. Explants of rat femoral fracture callus from days 4, 7, 14 and 28 post fracture induced NO2 release and ALP activity in a biphasic temporal manner, with the highest activity on day 7 and the lowest activity on day 14. Inhibition of NOS by co-incubation with an NOS inhibitor, S-(2-aminoethyl) isothiouronium bromide hydrobromide (AETU), inhibited ALP activity by an average of 50% at each time point (P <0.01). Supplementation with NO donor 3-morpholinosydnonomine hydrochloride (SIN-1) at low doses (25 and 0.025 microM) increased ALP activity by 20% (P < 0.01). ALP mRNA and histochemical ALP activity were localised to osteoblast-like and chondrocyte-like cells within fracture callus. The current study provides evidence that NO plays a regulatory role in ALP activity during rat fracture healing.

Differential potency of beclomethasone esters in-vitro on human T-lymphocyte cytokine production and osteoblast activity.

Beclomethasone dipropionate is an inhaled corticosteroid, used for the treatment of asthma. It is metabolised to 17-beclomethasone monopropionate, which has greater affinity for corticosteroid receptors than the parent compound, and to beclomethasone. We investigated the potency of beclomethasone dipropionate, 17-beclomethasone monopropionate and beclomethasone (compared with dexamethasone as a reference steroid) in two different human cell types, peripheral blood mononuclear cells and osteoblasts. We found that beclomethasone dipropionate, 17-beclomethasone monopropionate (EC50 10(-14) M) and beclomethasone (EC50 approx. 10(-12) M) were much more potent than dexamethasone (EC50 10(-8) M) in inhibiting interleukin-5 production by peripheral blood mononuclear cells. In contrast, beclomethasone dipropionate, 17-beclomethasone monopropionate and beclomethasone were equipotent with dexamethasone (EC50 range 0.3-1.2 x 10(-9) M) in affecting several functional assays of osteoblasts (e.g. alkaline phosphatase activity and osteocalcin synthesis). These results show that the relative bioactivities of corticosteroids vary between different human cell types, and that affinities observed in receptor binding assays are not necessarily predictive of the bioactivity in cell populations, such as peripheral blood mononuclear cells and osteoblasts, which are putatively relevant to efficacy and side effects respectively.

Comparative effects of anti-inflammatory corticosteroids in human bone-derived osteoblast-like cells.

While effects of inhaled corticosteroids on serum markers of bone metabolism in normal and asthmatic subjects have been reported, there are little data on the direct effects of these corticosteroids on end-organs such as bone. The results presented here compare the effects of budesonide and its epimers (22S- and 22R-budesonide), fluticasone and dexamethasone on growth and differentiation of cultured human bone cells. Osteoblast-like cells were cultured from human foetal bone chips grown to confluence and used at first subculture. At concentrations of 10(-11)-10(-7) M each corticosteroid (CS) caused a dose-dependent decrease in [3H]thymidine incorporation into deoxyribonucleic acid (DNA), median effective concentration (EC50): fluticasone (0.06 nM) >22R (0.26 nM) >22S (0.4 nM) >budesonide (0.47 nM) >dexamethasone (1.5 nM). Each CS resulted in a dose-dependent increase in alkaline phosphatase activity, EC50: fluticasone (0.14 nM) >22R (0.2 nM)=22S (0.2 nM) >budesonide (0.4 nM) >dexamethasone (1.6 nM). The 1,25 dihydroxyvitamin D3 (1,25(OH)2D3)-stimulated osteocalcin production was decreased in the presence of each CS, EC50: fluticasone (0.02 nM) >22S (0.1 nM) >22R (0.2 nM) >budesonide (1.0 nM) >dexamethasone (1.8 nM). In human bone cells the potencies of fluticasone and budesonide in relation to dexamethasone are not dissimilar to those derived from human lymphocytes in vitro.