Jean E. Russell

Bone deficit in ovariectomized rats. Functional contribution of the marrow stromal cell population and the effect of oral dihydrotachysterol treatment.

This study investigates the proliferative and osteogenic role of marrow stromal/osteoprogenitor cells in the development of the cortical bone deficit in ovariectomized (OVX) female rats. In vitro, clonal growth of marrow stromal cells from OVX rats was significantly impaired (vs. sham-operated controls). Yet in vivo, cells from sham-operated and OVX rats had equal osteogenic potential in several in vivo experimental situations, such as in intraperitoneally implanted millipore diffusion chambers and in intramuscular implants of marrow plus osteoinductive bone matrix (composite grafts). Long-term (6 mo) dihydrotachysterol (DHT) treatment of OVX rats enhanced their in vitro proliferative potential and clonal growth, as well as their osteogenic expression in composite grafts. The observation that the in vivo osteogenic performance of OVX rat marrow stromal cells was normal at extraosseous sites suggests that the mechanisms leading to osteopenia may involve an abnormality in cell-matrix interactions.

Effect of the Foot on the Mechanical Alignment of the Lower Limbs

The authors evaluated the effect of the foot on the loading axis of the lower limb measured from radiographs in 30 pediatric patients. Deviation at the knee was calculated for the hip-ankle (traditional) and the hip-foot lines (heel lined up with a metal wire). A trigonometric model of the limb loading axis was developed with predicted mechanical axis deviations at the knee. Statistics were based on the methods of Bland and Altman. Mechanical axis deviation at the knee in the frontal plane varies with foot height, foot-tibial angle, and genu valgum. The predicted trigonometric model was found to be in agreement with measured radiographic values. Including the foot in the radiographic measurement of limb alignment may increase validity of surgical planning for correction of malalignment and for evaluation of degenerative arthritis risk at the knee level.

Abnormal Bone Mineral Maturation in the Chronic Uremic State

X-ray diffraction analysis of bone from chronically uremic but nonacidotic rats with normocalcemia and hyperphosphatemia revealed smaller apatite crystals and an increase in the X-ray amorphous mineral fraction when compared to age-matched, pair-fed control animals, indicating less advanced mineral maturation in the uremic animals. Studies in animals with varied degrees of chronic renal insufficiency revealed a progression of the bone crystal maturational defect with advancing uremia.

Mineralization in rat metaphyseal bone exhibits a circadian stage dependency.

Abstract Density gradient fractionation analysis of rat metaphyseal bone was used to delineate the biorhythmic changes in bone matrix mineralization. Seventy-two 4-week-old rats were entrained to 12-hr light, 12-hr dark cycles (light, 0800-2000 hr; darkness, 2000-0800 hr) for 4 weeks. All animals were fed ad lib. on Purina laboratory rat chow and tap water. Groups of 10-12 rats were killed by cervical dislocation at 4-hr intervals during a 24-hr period, and the tibias were then biopsied and frozen in liquid N2. Metaphyseal bone was fractionated via bromoformtoluene density gradients into specific gravity fractions ranging from 1.7 to 2.8. Density gradient fractions were analyzed for concentrations of calcium and inorganic phosphorus. Chronograms indicated that the accumulation of both calcium and inorganic phosphorus into the newly forming/least-dense mineral moieties of bone (1.3-1.7 sp grav) showed a single peak in the biorhythm of the rat. A statistically significant circadian rhythm of mineralization was detected for calcium (P < 0.001) and inorganic phosphorus (P < 0.039), with peaks during the environmental dark span. These results suggest that the physiological phasing of bone mineralization in the light-dark synchronized rat, is similar to that previously noted for cartilage mineralization and is antiphasal to the midday peak in bone collagen synthesis.

25-hydroxycholecalciferol-enhanced bone maturation in the parathyroprivic state.

In vitro evidence presently favors a direct osteolytic effect of biologically active vitamin D metabolites. Studies were designed to evaluate the effect of 25-hydroxycholecalciferol (25OHD3) on bone collagen and mineral maturation in vivo and its dependence on parathyroid hormone (PTH). After treatment of sham-operated control and parathyroidectomized (PTX) mature rats with either 25OHD3 or an oil vehicle for 2 wk, tibial bone mineral-collagen maturation was quantitated by bromoform-toluene density gradient fractionation techniques. Intestinal calcium absorption was measured by in vivo 45Ca transport procedures. In contrast to the control group, the response to 25OHD3 of PTX rats was dramatic. Bone mineral and matrix maturation were both accelerated by 25OHD3 treatment without concomitant reduction in total bone mineral or collagen content or changes in the intestinal calcium absorption. These observations support the premise that biologically active vitamin D metabolites stimulate bone tissue maturation, and that PTH is not required in this regard.

The effect of protracted tetracycline treatment on bone growth and maturation

Mature female rhesus monkeys were used to evaluate the effects of a one-year course of tetracycline (50 mg/kg/day, intramuscularly) on the formation, maturation, and mineralization of mandibular bone. The bones from the treated group contained normal concentrations of calcium (Ca), inorganic phosphorus (Pi), and hydroxyproline (HO-Pr), and the treatment schedule did not alter the distribution (percentage) of total osteons into slightly, moderately, and highly mineralization classes. Tetracycline impairs bone mineralization and the subsequent maturation of the mineral and matrix moieties. The percentage of highly mineralized osteons labeled with tetracycline is subnormal. Density gradient fractionation studies indicate the presence of abnormally high Ca/Pi ratios in the temporally young newly formed bone mineral and somewhat higher ratios in the most mature bone fraction. Protracted tetracycline treatment at high dosages impairs bone growth and maturation in adult rhesus monkeys.

Effect of sodium fluoride on bone density in chickens

SummaryIn addition to increasing bone volume, fluoride has been demonstrated to increase ash weight and mineral density. To determine whether newly formed or older bone is most affected by fluoride treatment, bone from chickens receiving fluoridated water was fractionated into lower density (recently formed) and higher density (more mature) specific gravity fractions. Fluoride was administered to the chickens for different lengths of time (4 or 13 weeks) or at varying doses for a 4-week period (0, 4.2, 16.8 mmol/liter drinking water). Fluoride treatment caused a shift in the mineral density profile, showing an increased proportion of mineral distribution in the more mature, higher density fractions.To determine whether this density gradient shift was due to increased maturation rate of bone or decreased resorption and mineralization rates, [3H]proline and 45Ca were injected 5 days and 24 hours prior to sacrifice, respectively. The distributions of both 3H or 45Ca, as percentages of total counts incorporated, were shifted by fluoride treatment into more mature, higher density fractions. Expressing the number of counts as a percent of the bone in each fraction (total hydroxyproline or Ca) revealed an increased incorporation of both 3H and 45Ca into the higher specific gravity fractions 2.0–2.2. These results suggest that fluoride treatment increases bone maturation and the rate of secondary mineralization in the cortical bone. Such changes in the quality of more mature, well-mineralized bone, in humans as well as animals, may have a significant influence on brittleness and strength.

Effect of an adrenocorticotropin analogue, ACTH 1-17, on DNA synthesis in murine metaphyseal bone

The effects of injections of a synthetic adrenocorticotropin (ACTH 1-17, Synchrodyn) on the rate of DNA labeling in the metaphyseal bone of CD2F1 mice were tested on a chronopharmacological dosing schedule. Groups of mice that had been conditioned to a 12-hr light/12-hr dark schedule were injected at one of six different timepoints, 4 hr apart, during a single 24-hr span with either a low (0.02 I.U./kg) or a high (20 I.U./kg) dose of ACTH 1-17. Control groups received injections of a placebo at corresponding timepoints. Subgroups of mice were injected with [3H]thymidine ([3H]Tdr) to follow the changes in DNA labeling in the proximal tibial metaphysis at 15 min and 2, 4, 8, 12 and 24 hr after ACTH 1-17 or placebo treatment. All mice were injected with the isotope 30 min before killing, except for those killed 15 min after Rx administration where the isotope had been injected 14 min before killing. The data were analyzed both by analysis of variance and by the cosinor method, the latter of which tests the fit of a 24-hr cosine curve to the data. The effect of ACTH 1-17 on the target cell population was dependent not only upon the dose but upon the time of administration. Both doses exerted time-dependent action, ranging from stimulation to inhibition of DNA labeling. Inhibition was noted when the ACTH 1-17 was administered at 2 hr after the beginning of the daily dark span when nocturnal animals become active. When administered at this circadian stage, the larger dose in particular was associated with an inhibition of DNA labeling lasting for 24 hr. The inhibitory effect was much shorter when the same dose was injected 4 hr earlier. Moreover, the large ACTH 1-17 dose had a stimulatory effect lasting for 24 hr when it was administered 2 hr after the onset of the daily light span, with a much shorter stimulation following administration of the large dose at 6 hr after the beginning of the daily dark span. A circadian stage-dependent stimulation or inhibition of DNA labeling at 2 or 14 hr after light onset, respectively, was thus complemented by an initial inhibition followed by stimulation and vice versa at 10 and 18 hr after light onset respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Experimental renal osteodystrophy. The response to 25-hydroxycholecalciferol and dicholomethylene diphosphate therapy.

Bone mineral and matrix maturation in chronically uremic, nonacidotic rats were investigated after 25-hydroxcholecalciferol (25OHD) and/or dichloromethylene diphosphonate (C12MDP) therapy utilizing bromoform-toluene density gradient fractionation and X-ray diffraction analyses. The bromoform-toluene density gradient analyses demonstrated that the progressive accumulation of less dense, more immature bone characteristic of progressive uremia was reversed by 25OHD and/or C12MDP therapy for a 2-wk period, and that after 4 wk of therapy the maturational profile of bones from chronically uremic animals treated with 250HD and/or C12MDP was comparable to that from nonuremic littermates. X-ray diffraction analysis revealed that by the 4th wk of therapy with 25OHD and C12MDP both the degree of crystallinity and the crystal size/perfection parameters in the uremic bones were comparable to those of nonuremic, pair-fed control littermates. Treatment for 4 wk with 25OHD resulted in enlarged and/or more perfect apatite crystallites, while C12MDP alone slightly inhibited crystal growth and/or perfection after 2 wk of treatment. Soft tissue calcification was diminished in uremic animals treated for 4 wk with C12MDP or a combined C2MDP/25OHD regimen, the latter being much more effective in this regard. The accumulated data in this study support the premise that the attendant accelerated bone resorption, soft tissue calcification, and abnormal mineralization and maturation of the skeletal tissue, well documented to characterize experimental ranal insufficiency, may be alleviated with therapeutic dosages of 25OHD and/or C12MDP.

Regional differences in matrix formation in the healing flexor tendon.

In 20 white leghorn chickens, the effect of proximity (proximal vs. distal) to the repair site and the effect of sheath excision versus sheath repair on biochemical matrix formation in the healing zone 2 flexor tendon were studied. In 14 animals, the profundus tendon of the long toe on both feet was lacerated and repaired in zone 2. In the right foot, the sheath was repaired, and on the left foot, the sheath was excised. Segments proximal and distal to the laceration site were studied at six weeks postrepair compared to control segments. Sheath excision versus sheath repair had no effect on the net matrix formation of the healing flexor tendon. A higher DNA content and lower hydroxyproline and hexosamine contents were present in the healing tendons, indicating the healing process was not complete at six weeks. Sheath excision versus sheath repair had no effect on net matrix formation in the healing tendon. There is no difference in DNA content or glycosaminoglycan content in the proximal versus distal segments. There was a significantly greater net hydroxyproline content in the proximal segment versus the distal segment of the healing tendon, which was not present in similar control segments. These results support the concept that the zone 2 flexor tendon does not respond to injury as a homogeneous structure.