H. K. Väänänen

The mechanical strength of bone in different rat models of experimental osteoporosis

In order to discover good parameters for experimental osteoporosis, we measured the failure load of the femoral neck and the bending strength of the tibia in orchidectomized (ORC) (20 rats for 4 weeks), ovariectomized (OVX) (28 rats for 6 weeks), and immobilized (IMM) (33 rats for 3 weeks) rats. Each of these operations led to a significant decrease in trabecular bone volume when compared with corresponding controls (p < 0.001). The ash weight of femurs was significantly decreased in ORC (p < 0.05) and IMM (p < 0.001) rats, but not in OVX rats. Growth of the femur was somewhat slower in ORC (p < 0.05) and IMM rats (p < 0.05), but not in OVX animals. All three osteoporosis models showed significant decreases in the maximal load of the femoral neck (ORC: 23.9%, p < 0.001; OVX: 15.8%, p < 0.001; IMM: 27.7%, p < 0.001), as well as in energy absorption (ORC: 43.9%, p < 0.001; OVX: 28.3%, p < 0.001; IMM: 45.3%, p < 0.001). In tibia orchidectomy reduced maximal strength and energy absorption significantly (10%, p < 0.01; 27.8%, p < 0.01), but ovariectomy decreased only maximal load (8.7%, p < 0.01) and immobilization only energy absorption (18.0%, p < 0.01). Our results suggest that the mechanical strength of the femoral neck is a sensitive indicator of bone loss in all three osteoporosis models.

Omeprazole, a specific inhibitor of H + −K + -ATPase, inhibits bone resorption in vitro

SummaryOmeprazole has been previously shown to block gastric acid secretion by specific inhibition of gastric parietal cell membrane H+−K+-ATPase. It is now demonstrated that similar concentrations of omeprazole will inhibit PGE2- and PTH-stimulated Ca++ release from prelabelled neonatal mouse calvariae without affecting the viability of cultured calvaria explants.

The effects of bisphosphonates on the resorption cycle of isolated osteoclasts

Binding sites for wheat germ agglutinin (WGA)-lectin have been shown to become revealed in the demineralized resorption lacunae that osteoclasts excavate on bone substrate. Peroxidase-conjugated WGA-lectin, which binds to bone matrix glycoconjugates and proteoglycans, was used in pit formation assays to assess the activity of isolated osteoclasts cultured on either 3-amino-1,1-hydroxy-propylidene-bisphosphonate (APD)-or dichloromethylene bisphosphonate (Cl2MBP)-covered bone slices. Immunofluorescence and histochemical techniques were also used to study the effects of bone-bound bisphosphonates on isolated rat osteoclasts. Neither APD nor Cl2MBP interfered with the special organization of actin or vinculin in osteoclasts when the cells were initializing their resorption cycle. After 24 hours of culture, the number of resorbing osteoclasts increased strongly on control slices, but remained low on either APD- or Cl2MBP-treated slices. At this time, the actin and vinculin rings in osteoclasts also started to exhibit abnormal, more diffuse staining. Both bisphosphonates studied resulted in signs of cytotoxicity: the number of osteoclasts decreased on APD- or Cl2MBP-covered bone during the course of the study and those remaining attached exhibited severe cytoplasmic retractions. The total areas of resorption remained at significantly lower levels in both experimental groups studied, and this was due to decreases in both the number and sizes of individual resorption pits. The size of the most extensive lacunae detected on the Cl2MBP slices did not exceed 5x103 μm2, whereas on the control slices, resorption pits bigger than 15x103 μ2 were frequently discovered.

β1 integrins and osteoclast function: Involvement in collagen recognition and bone resorption

Abstract The extracellular matrix of bone is composed mainly of type I collagen. In this report we studied the role and collagen binding properties of osteoclast integrins ( α v , α 2 , β 1 , and β 3 ). Cell adhesion assays with rat osteoclasts and affinity chromatography/SDS-PAGE analysis with purified human osteoclast membranes demonstrated adhesion of osteoclasts to native type I collagen in a divalent cation and Arg-Gly-Asp (RGD)-dependent way via α 2 β 1 integrin, whereas osteoclast adhesion to denatured collagen predominantly involved α v β 3 . In receptor-binding assays, the involvement of human recombinant α v β 3 in adhesion to denatured collagen was confirmed. Additionally, osteoclasts adhered to type I collagen fibers and to monomeric types II–V collagen with characteristics similar to those on native monomeric type I collagen. ] Osteoclastic bone resorption in vitro was inhibited (>40%) in the presence of α2 and β 1 antibodies. Using scanning laser confocal microscopy, α v β 3 , α 2 , and β 1 integrin were detected within podosomes in nonresorbing osteoclasts and in the ruffled border area and basolateral membrane in resorbing osteoclasts, but not in the sealing zone of resorbing osteoclasts. These results demontrate that α 2 β 1 , in addition to α v β 3 , has an important role in osteoclast function and acts as a receptor for native, but not denatured, collagen.

Long-term effects of ovariectomy on the mechanical properties and chemical composition of rat bone

One hundred Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (Sham) at the age of 12 weeks. Eight or six animals from each group were sacrificed at the following time points: 0, 2, 4, 8, 16, 28, and 40 weeks. Mechanical testing of the bones showed that the maximal load of the femoral neck remained at the baseline level in OVX rats but clearly increased in Sham animals. However, after 28 weeks (at the age of 40 weeks) of ovariectomy, the femoral neck strength in Sham animals started to decrease, reaching the OVX level at the age of 1 year. The gain of torsional strength of the humerus was first slowed down after ovariectomy, but 16 weeks after operation this difference between the OVX and Sham animals disappeared. In OVX rats incorporation of 45 Ca2+ into the tibia and the number of osteoclasts on the trabecular bone surfaces were increased at 4-16 weeks and at 2-28 weeks, respectively, but they returned later to the levels observed in Sham animals. This indicates that the OVX-induced increase in bone turnover is transient. The trabecular bone volume at the distal femur decreased and the area of the marrow cavity in the distal third of the femur increased after OVX and did not show any recovery. Analysis of the inorganic composition of bone by energy-dispersion spectrometry showed that the percentage of magnesium was transiently decreased after OVX in both trabecular and cortical bone. Despite the OVX-induced loss of trabecular bone, increasing body weight or some metabolic changes may induce structural changes which may be responsible for maintaining the mechanical force of the femoral neck in aging OVX rats.

Ovariectomy-Induced Bone Loss Can be Affected by Different Intensities of Treadmill Running Exercise in Rats

Abstract. Fifty-six Sprague-Dawley rats were either ovariectomized (OVX, n= 24), sham-operated (Sham, n= 24), or sacrificed (n= 8) at the beginning of the experiment to serve as a baseline group. The OVX and Sham groups were further randomly divided into control (CTRL), slow running (R10), and faster running (R18) groups. R10 and R18 groups ran for 2 × 30 min/day for 8 weeks at speeds of 10 m/min and 18 m/min, respectively. Exercise did not affect the mechanical or histomorphometric parameters of bone in the sham-operated rats. There was no effect of exercise on body weight gain in the OVX-R10 group, but in OVX-R18 it decreased the gain of body weight. In the OVX–CTRL group the maximal load and energy absorption of the femoral neck were 16.7% (P < 0.001) and 30.0% (P < 0.001) lower than in the Sham–CTRL group, respectively. In OVX animals, slow running had a positive effect on the maximal load of the femoral neck (86.5 N) when compared with OVX–CTRL rats (77.1 N, P < 0.07). 51.7% of the trabecular bone was lost in the distal femur as a result of OVX and exercise reduced this loss to 30.2% (R10) and 39.9% (R18). Ovariectomy increased the bone formation rate (BFR) and the mineral apposition rate (MAR) on the periosteum of the femoral shaft. Exercise decreased the periosteal BFR and MAR in OVX rats, but increased it at the endosteum. Osteoclast numbers in the femoral metaphysis were increased after OVX and running exercise inhibited this effect significantly. The maximal bending load of the humerus increased after OVX by 12.1% (P < 0.05). Exercise enhanced this effect, the slow running being more effective. These results suggest that bone in OVX rats is either more sensitive to exercise than in sham-operated rats or that the higher body weight with slow running induces optimal loading and strengthens the bones.

Carbonic anhydrase isoenzymes in isolated rat peripheral monocytes, tissue macrophages, and osteoclasts

The presence of carbonic anhydrase isoenzymes I and II in rat monocytes and macrophagelike cells was studied using monospecific antisera against rat carbonic anhydrase I and II purified from red blood cells. CA II was strongly stained immunohistochemically in osteoclasts and macrophagelike cells in the umbilical cord. Foreign body giant cells, peritoneal macrophages, lung macrophages, and cultured peripheral monocytes, the presumed progenitor cells for osteoclasts, were negative with both antisera. Radioimmunoassay and immunoblotting similarly failed to demonstrate CA II in peripheral monocytes. The lack of CA in monocytes adds a new aspect to the discussion concerning the origin of osteoclasts and monocyte-mediated bone resorption.

Effect of running exercise on the bone loss induced by orchidectomy in the rat

The effect of exercise on castration-induced osteoporosis in 3-month-old male rats weighing 264±4 g at the beginning of the experiment was studied. A testosterone deficiency was induced by orchidectomy (ORC), and the exercise group ran 10 m/minute for 1 hour a day on a treadmill at 0% grade. There were seven groups of eight rats (n=56) randomized into a control group killed at time 0, and sham, ORC and ORC and exercise groups killed at 4 and 8 weeks. ORC reduced body weight gain (with analysis of variance (ANOVA) P<0.001), and at 4 weeks the body weight was 343±14 g in ORC group and 301±4 g in the ORC and exercise group (P<0.01). The increase in femoral length was slower in the ORC+exercise groups. The ash weight of the tibia did not decrease significantly after ORC or ORC+ exercise. ORC did not affect 45Ca incorporation, but exercise slightly increased it in the whole tibia 8 weeks after ORC (with ANOVA P=0.057). ORC had significantly lowered the trabecular bone volume in the secondary spongiosa of the distal femur at 4 and 8 weeks, and exercise did not prevent this. This is an opposite finding to our previous study with ovariectomized female rats [12]. ORC also significantly had reduced the osteoblast-lined trabecular bone surface and the number of osteoclasts by 8 weeks after the operation. Exercise increased the osteoblast-lined surface and the number of osteoclasts. The mechanical strength of the femoral neck also was reduced after ORC and this was not prevented by exercise either. In conclusion, ORC reduces bone growth and turnover which leads to osteopenia in growing rats. Moderate treadmill exercise does not reverse the ORC-induced loss of trabecular bone and the reduced mechanical strength of the femoral neck, although it has a positive effect on the osteoblast and osteoclast indices and on calcium incorporation into bone.

Long-term effects of clodronate on growing rat bone.

For evaluating the long-term effects of the bisphosphonate compound clodronate on the rat skeleton, 100 female rats were given subcutaneous injections of clodronate at doses of 0 (vehicle), 4 (low), or 12 (high) mg/kg per week, or 50 mg/kg every fourth week (cyclical). The treatment was started at 3 months of age and was continued for 6 months. The mechanical strength of bones was studied by torsion of the tibia, three-point bending of the femur, axial compression of the femoral neck, and compression of a lumbar vertebra. Quantitative histomorphometric variables were determined from distal femur and lumbar vertebra, and variables reflecting bone metabolism were measured in serum and urine. Bone mass, indicated by ash weight of the tibia, was increased with the low and high clodronate doses compared with the vehicle. The maximum load in vertebra compression was increased with the low dose of clodronate compared with the vehicle, whereas changes in other variables concerning bone strength were not significant. In bone histomorphometry, clodronate treatment induced more pronounced changes in cancellous bone volume in distal femur than in lumbar vertebra, the differences not being statistically significant between the groups at either site, however. The longitudinal growth rate of the femur, measured by double-fluorochrome labeling for 1 week at the end of the treatment period, was significantly decreased in the high-dose clodronate group compared with the other groups. Serum values for calcium, tartrate-resistant acid phosphatase, and alkaline phosphatase did not differ between the groups. However, serum osteocalcin was significantly lower in the high-dose group compared with the vehicle group. Urinary calcium, hydroxyproline, and hydroxylysylpyridinoline were decreased at all the clodronate doses administered. In conclusion, the beneficial effects of long-term clodronate treatment on bone mass and strength were observed at the lowest dose used. A high dose of clodronate decreased bone growth rate, which was, however, not reflected in the mechanical quality of bone.

Immunohistochemical demonstration of extracellular carbonic anhydrase in epiphyseal growth cartilage

SummaryThe distribution of carbonic anhydrase isoenzymes C and B in the rat epiphyseal growth cartilage was demonstrated by an immunohistochemical method. The isoenzymes were found in different locations. Isoenzyme C was in the extracellular matrix of the hypertrophic and calcifying cartilage, and no reaction was observed in the chondrocytes. In contrast, the antiserum against isoenzyme B revealed only a weak cellular staining. This supports the hypothesis that carbonic anhydrase isoenzyme C, which is the high-activity form, changes the pH in the extracellular fluid of calcifying cartilage, favoring the deposition of calcium phosphate.