Comparative high-resolution pQCT analysis of femoral neck indicates different bone mass distribution in osteoporosis and osteoarthritis.

SUMMARY: Osteoarthritis is linked to a reduced risk of femoral fracture despite osteoporosis. Different bone distribution in the femoral neck in osteoarthritis and fracture was revealed using a peripheral quantitative computed tomography (pQCT) comparative analysis. Our findings sustain the presence of an adaptive mechanism of bone structure providing fracture protection in osteoarthritis. INTRODUCTION: Although osteoarthritis is associated with reduced femoral fracture risk, it does not protect from bone loss. We investigated whether adaptive mechanisms are present at the arthritic joint, leading to reduced fracture risk, despite the presence of low bone mass density. METHODS: We performed pQCT comparative analyses of human femoral neck specimens derived from 32 postmenopausal women who received hip prostheses for osteoarthritis (n = 19) or femoral fracture (n = 13) by applying an in-house automated software to extract bone structure descriptors, characterize trabecular and cortical bone distribution, and evaluate their mutual relationships. RESULTS: The cortical bone volume and trabecular thickness were significantly (p < 0.05) higher in the osteoarthritis group than in the fracture group. Trabecular bone volume was also significantly (p < 0.05) higher in the osteoarthritis group than the fracture group at the inferior and anterior quadrants. Significance was maintained after adjusting for age, cortical bone volume, and cortical porosity thickness. Multiple linear regression analysis showed that thickness, volume, and apparent density of the trabecular region significantly (p < 0.05) correlated with the same cortical descriptors in osteoarthritis, but no significant relationship was found in the fracture group. Age differentially affected the mutual relationships in the two groups, showing a significant correlation with trabecular thickness in both groups and with apparent trabecular density only in femoral fracture group. CONCLUSIONS: Starting from these differences in the structural descriptors, our study sustains the presence of a compensatory mechanism in osteoarthritis to preserve the mechanical competence of bone structure, despite the loss of trabecular bone, underlying lower fracture risk.

A preliminary study of the use of peripheral quantitative computed tomography for investigating root canal anatomy.

AIM: To evaluate the use of peripheral quantitative computed tomography (pQCT) for qualitative and quantitative analysis of root canal anatomy and for assessing the extent of canal enlargement during root canal instrumentation. SUMMARY: The volume variation achieved by S1 ProTaper instruments in the coronal third of the root canals was analysed using peripheral computed tomography. The tooth was scanned in the horizontal plane producing 36 consecutive cross-sectional images. All images were the result of 360 projections with a section thickness of 250 microm, a distance between slices of 0.5 mm and an in-plane pixel size of 70 x 70 microm. The evaluation was completed before and after S1 ProTaper instrumentation (with or without circumferential filing) of one root canal of a freshly extracted maxillary first premolar tooth. The acquired images were realigned geometrically and processed using a 3D visualization software. pQCT scanning allowed 3D reconstruction of the root canal anatomy and the assessment of the extent of canal enlargement during root canal instrumentation with lateral displacement of canal walls and hence volume change being greater than the coefficient of variation. The densitometry evaluation showed uniform density along the root canal wall. KEY LEARNING POINTS: * pQCT scanning allowed 3D reconstruction of the root canal anatomy and the assessment of the extent of canal enlargement during root canal instrumentation. * pQCT shows promise for allowing qualitative and quantitative analysis of endodontic procedures.

Sympathectomy alters bone architecture in adult growing rats.

Sympathetic nervous system (SNS) fibres and alpha- and beta-receptors are present in bone, indicating that the SNS may participate in bone metabolism. The importance of these observations is controversial because stimulation or inhibition of the SNS has had various effects upon both anabolic and catabolic activity in this tissue. In this study we evaluated the effects of pharmacological sympathectomy, using chronic treatment of maturing male rats with 40 mg of guanethidine/kg i.p., upon various parameters in bone. Double labelling with tetracycline injection was also performed 20 and 2 days before sacrifice. Bone mass, mineral content, density and histomorphometric characteristics in different skeletal regions were determined. Bone metabolic markers included urinary deoxypyridinoline and serum osteocalcin measurements. Guanethidine significantly reduced the accretion of lumbar vertebral bone and of mineral content and density, compared to controls. Femoral bone mineral content and density were also significantly reduced, compared to controls. Histomorphometric analyses indicated these effects were related to a reduction of cortical bone and mineral apposition rate at femoral diaphysials level. Both markers of bone metabolism were reduced in controls as they approached maturity. Guanethidine significantly decreased serum osteocalcin compared to controls, while urinary deoxypyridinoline was unchanged. These data indicate that guanethidine-induced sympathectomy caused a negative balance of bone metabolism, leading to decreased mass by regulating deposition rather than resorption during modeling and remodeling of bone.

Growth hormone stimulates osteoprotegerin expression and secretion in human osteoblast-like cells.

It is presently thought that osteoprotegerin (OPG) is a cytokine involved in the regulation of osteoblast/osteoclast crosstalk and maintenance of bone mass. Recent studies showed that GH replacement therapy in GH-deficient patients was able to induce a significant increase of OPG in the plasma, as well as in the cortical and the trabecular bone. In order to determine whether GH could directly modulate OPG secretion, the effect of GH on human osteoblast-like cells (hOB) in primary culture was studied. After detecting the presence of the mRNA for the GH receptor (GHR) by RT-PCR, hOB were exposed to increasing concentrations of GH, from 0.1 to 25 ng/ml, for 24 h. The results showed that GH exposure was able to stimulate OPG secretion in a concentration-dependent manner. In addition, the OPG mRNA levels were increased, indicating that the hormone has a stimulatory effect on gene expression. The stimulatory effect on OPG expression and production was prevented by exposing the cells to tyrphostin AG490 (10 muM), an inhibitor of Janus kinase 2, which is one of the kinases involved in the intracellular pathway activated by the binding of GH to its receptor. Similar results were obtained when the cells were exposed to a receptor antagonist of GH, pegvisomant at 50 nM. GH exposure neither induced an increase in IGF-I expression nor secretion in hOB. These results suggest that the stimulation of OPG production induced by GH in hOB is specific and receptor mediated and further support the view that GH is able to modulate bone remodeling by directly influencing osteoblast-osteoclast crosstalk.

Different skeletal regional response to continuous brain infusion of leptin in the rat.

This study was designed to evaluate whether or not continuous intracerebroventricular infusion of leptin (1.5 microg/rat/24 h, for 28 days) produced different regional response on the skeleton of growing rats. Leptin reduce the accretion of total femoral bone mineral content (BMC) and density (BMD). This effect was related to a reduction of metaphyseal femur as no changes were detected in the diaphysis. Despite the reduced accretion in the volumetric of both femur and tibia compared to controls, leptin had no significant effects on the lumbar vertebrae. Urine deoxypyrydinoline and serum osteocalcin remained more elevated in the leptin-treated group as compared to controls. The results demonstrate that long-term central infusion of leptin activates bone remodeling with a negative balance. Leptin induces distinct responses in the different structure of bone and in the axial and appendicular skeleton.

Bone as an ion exchange organ: evidence for instantaneous cell-dependent calcium efflux from bone not due to resorption.

The current study tests the hypothesis that basal level and minute-by-minute correction of plasma Ca2+ by outward and inward Ca2+ fluxes from and into an exchangeable ionic pool in bone is controlled by an active partition system without contributions from the bone remodeling system. Direct real-time measurements of Ca2+ fluxes were made using the scanning ion-selective electrode technique (SIET) on living bones maintained ex vivo in physiological conditions. SIET three-dimensional measurements of the local Ca2+ concentration gradient (10 microm spatial resolution) were performed on metatarsal bones of weanling mice after drilling a 100-mum hole through the cortex to expose the internal bone extracellular fluid (BECF) to the bathing solution, whose composition mimicked the extracellular fluid (ECF). Influxes of Ca2+ towards the center of the cortical hole (15.1+/-4.2 pmol cm-2 s-1) were found in the ECF and were reversed to effluxes (7.4+/-2.9 pmol cm-2 s-1) when calcium was depleted from the ECF, mimicking a plasma demand. The reversal from influx to efflux and vice versa was immediate and fluxes in both directions were steady throughout the experimental time (>or=2 h, n=14). Only the efflux was nullified within 10 min by the addition of 10 mM/L Na-Cyanide (n=7), demonstrating its cell dependence. The timeframes of the exchanges and the stability of the Ca2+ fluxes over time suggest the existence of an exchangeable calcium pool in bone. The calcium efflux dependency on viable cells suggests that an active partition system might play a central role in the short-term error correction of plasma calcium without the contribution of bone remodeling.

Application of high resolution pQCT analysis for the assessment of a bone lesion: a technical note.

Peripheral quantitative computed tomography (pQCT) has found new fields of application in bone medicine, but none of them concerns the forensic practice. This study exposes the potential of pQCT applied to a penetrating lesion in a vertebral body. A pQCT scanner was used for the measurements (XCT Research SA+; Stratec Medizintechnik GmbH, Pforzheim, Germany). A more precise reconstruction of the path of the lesion within the trabecular bone was reached, with more details concerning the morphological characteristics of the lesion inside the vertebral body, and the elaboration of a 3D model was created, which allowed the operator to define the volume of the lack of tissues related to the lesion. The application of pQCT scan proved to be a potentially useful tool for the assessment of bone lesions, although further studies are needed in order to verify its applicability to forensic context.

Comparison between urinary pyridinium cross-links and hydroxylysine glycosides in monitoring the effects of ovariectomy and 17 beta-estradiol replacement in aged rats.

This study was undertaken to assess the sensitivity of hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), galactosylhydroxylysine (GHyl) and glucosylgalactosylhydroxylysine (GGHyl) to monitor bone response to estrogen deficiency and replacement by comparing their excretory patterns in ovariectomized aged (11-14 months old) rats. The ovariectomized (OVX) rats were randomized into two groups: (1) OVX plus vehicle; (2) OVX plus 17 beta-estradiol (17-beta E, 10 micrograms/kg, s.c., 4 days/week). Treatment with 17-beta E started immediately after OVX and continued for 60 days. The collagen catabolites were measured in urine for 1 month before OVX and thereafter for 60 days. In temporal coincidence with urine collection, bone area and bone mineral density (BMD) of lumbar vertebrae, femoral diaphysis and distal metaphysis were measured by dual-energy X-ray absorptiometry. In the untreated rats, BMD of the femoral metaphysis and lumbar vertebrae decreased significantly and the urinary excretion of LP, HP, GHyl and GGHyl increased with different patterns. In the treated rats, 17-beta E replacement prevented the increment in LP excretion, partially prevented the increase in HP excretion, but had no effect on the excretion of GHyl and GGHyl. In conclusion pyridinolines and glycosides have different sensitivities to the bone response to OVX. Glycoside excretion after OVX also reflects metabolic processes not strictly related to bone loss and, in contrast with LP, is not sensitive to estrogen replacement.

Red cell aging and active calcium transport.

The authors have investigated the relationships between the active calcium transport across the human red blood cell (RBC) membrane and the RBC aging processes in vivo and in vitro. For the study of this biological system, the authors have determined the active calcium uptake by inside-out membrane vesicles obtained from selected RBC populations. This model provided an optimal way to assess the biochemical and functional responses of the human cell to the oxidative stimulus triggered by the cellular aging processes. The activity of the calcium pump is indeed strictly correlated to the oxidative damage suffered by the RBC, being higher in the aged RBC. It appears that the main controller of the active calcium transport is the age-dependent protein inhibitor of the calcium pump.

Responsiveness of urinary markers of bone resorption to orchiectomy and clodronate treatment in mature rats: a comparative study.

OBJECTIVE: The aim of this study was to assess the responsiveness of two classes of bone resorption markers to the enhancement of osteoclastic activity induced by orchiectomy and to its inhibition by clodronate treatment in mature rats. DESIGN: Bone mineral density (BMD) at femural metaphysis, femural diaphysis, lumbar vertebrae, and the urinary excretion of pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosylhydroxylysine (GHyl) and glucosylgalactosylhydroxylysine (GGHyl) were monitored at regular intervals for 30 days prior to and for 60 days following orchiectomy in eleven rats, divided into two groups: five rats untreated and the other six treated with clodronate. RESULTS: Prior to orchiectomy, a significant (P < 0.01) decrease in BMD was observed only at the distal femural metaphysis. This decrease appeared to be associated with a time-dependent increase in the urinary excretion of all markers. Following orchiectomy, the BMD of the untreated group decreased significantly (P < 0.01) at all bone sites. The bone loss was accompanied by a significant (P < 0.01) increase in Pyr and D-Pyr concentrations in urine, whereas urinary GHyl and GGHyl did not change significantly. In the clodronate-treated group, the BMD of the three skeletal sites did not change significantly, while the urinary excretion of all urinary biochemical markers decreased significantly (P < 0.001). CONCLUSION: This study showed that pyridinolines are able to monitor the bone response to orchiectomy and to clodronate treatment response in androgen-deficient mature male rat. whereas glycosides appear prone to confounding factors.

Bone effects of hexarelin, a GH-releasing peptide, in female rats: influence of estrogen milieu.

OBJECTIVE: The present study was performed to evaluate the potential influence of the estrogen milieu in modulating the effects of GH/IGF stimulation by a GH-releasing peptide, hexarelin (HEXA), on bone metabolism and mineral density in middle-aged female rats. METHODS: HEXA was administered for 60 days (50 microg/kg s.c. twice a day) to intact and ovariectomized (OVX) 11-month-old female rats and changes in bone parameters were evaluated with respect to those of the same rats under baseline conditions and with those of control rats (intact and OVX) administered isovolumetric amounts of physiological saline. Serum total alkaline phosphatase (ALP) and urinary deoxypyridinoline (Dpd) were measured before and at various times during HEXA treatment. Bone mineral content (BMC) and density of lumbar vertebrae and femoral mid-diaphyses were measured by dual energy X-ray absorptiometry before and after treatment. In all groups, serum IGF-I levels were determined before and during treatment and the GH secretory response to HEXA was assessed at the end of the experiment. RESULTS: In intact rats, HEXA did not modify Dpd urinary excretion, induced a trend toward an increase of serum ALP activity and significantly increased BMC (+6.5%) and bone area (+4.1%) only at lumbar vertebrae. In OVX rats, HEXA did not modify the OVX-induced increase in bone turnover markers (Dpd and ALP) and did not affect the OVX-induced vertebral bone loss, but significantly increased BMC (+7.2%) and bone area (+5.3%) at femoral mid-diaphyses. HEXA significantly increased serum IGF-I levels at day 14, but not at day 60, in both intact and OVX rats, whereas the GH secretory response to HEXA was higher in the former than in the latter. CONCLUSIONS: Overall, the present data demonstrate that chronic HEXA treatment increases BMC and bone area at lumbar vertebrae in intact rats and at femoral diaphyses in OVX rats. The different sensitivity to HEXA of the skeletal districts examined is related to the estrogen milieu and may reflect a complex interplay between estrogens and GH/IGF function.

Effects of amylin on human osteoblast-like cells.

Amylin has been reported to have bone-conserving effects. In the present study we evaluated the possible activity of the peptide on human osteoblast-like (hOB) cells in primary culture. Amylin between 10(-9) and 10(-6) M, dose-dependently stimulated cell proliferation with a maximal effect (200%) at 10(-6) M. In addition, amylin increased osteocalcin production when hOB cells were exposed to 1,25(OH)2D3 (10(-8)M) but there was a nonsignificant upward trend on alkaline phosphatase activity. The present results suggest that amylin could be included among the group of peptides endowed with osteogenic activity.

Hexarelin, a growth hormone - releasing peptide, counteracts bone loss in gonadectomized male rats.

The age-related decline in growth hormone (GH) secretion has been implicated in the pathogenesis of involutional bone loss. Whether restoration of GH secretion might be helpful in maintaining and/or improving bone mass during aging is still unsettled. The aim of the present study was to examine the effects of 30-day treatment with hexarelin (HEXA, 50 microg/kg subcutaneously b.i.d.), a highly effective GH-releasing compound, on bone metabolism and bone mineral density (BMD) in intact and osteopenic gonadectomized (GDX) mature male rats. Serum total alkaline phosphatase (ALP, bone formation marker) and bone resorption markers (lysylpyridinoline, LP and hydroxylysylpyridinoline, HP) were measured before and 7, 14 and 30 days after treatment. BMD was measured by dual-energy X-ray absorptiometry at lumbar vertebrae, femoral metaphysis and diaphysis before and at the end of the experiment. In intact rats, HEXA significantly (P<0.05) decreased LP (-36.3%) and HP (-22.8%) excretion at day 7, whereas it did not change serum ALP activity and BMDs. In GDX rats, HEXA completely prevented the significant (P<0. 01) increase in urinary excretion of both LP (+143.8%) and HP (+119. 4%), the early decrease in ALP activity (-26.5%) and the significant (P<0.05) decrease in BMDs in the femoral metaphysis (-7.9%) and lumbar vertebrae (-6.8%) caused by androgen deficiency. The bone-protective effects of HEXA could be attributed, at least in part, to its GH-releasing activity since chronic-treated rats maintained the GH response to an acute challenge with HEXA. The evidence that HEXA, unlike GH, inhibits bone resorption indicates that other mechanisms contribute to the bone sparing effect of HEXA.

Effects of calcitonin gene-related peptide and amylin on human osteoblast-like cells proliferation.

Expression of mRNA for calcitonin gene-related peptide (CGRP) and CGRP receptor has been detected in osteoblasts indicating that CGRP could play a role in bone metabolism. In the present study, we evaluated the effect of CGRP on primary culture of human osteoblast-like cells proliferation. The peptide was able to stimulate [3H]thymidine incorporation in human osteoblast-like cells with a maximal effect at 10(-8) M. The proliferating activity of CGRP was not inhibited by the two antagonists, CGRP-(8-37) or amylin-(8-37), whereas amylin fragment antagonized the proliferating activity of amylin. In human osteoblast-like cells CGRP, but not amylin, was able to stimulate adenylyl cyclase activity and this effect was completely antagonized only by CGRP-(8-37) and not by amylin-(8-37). These data suggest that the CGRP induced stimulation of cAMP is not involved in the peptide proliferating effect in human osteoblast-like cells and that in this cell population there are receptor subtypes for CGRP, distinct from that of amylin.

Changes in bioelectric potentials on bone associated with direct current stimulation of osteogenesis.

This study was designed to determine whether changes occur in the bioelectric potentials on bone during and after bone stimulation with a 20-microA direct current (DC) and whether the variations in bioelectric potentials are related to the variations in bone formation. The bioelectric potentials were recorded at different times on the rabbit distal tibial surface, during (current-on state) and after (current-off state) DC stimulation with a cathode implanted within the medullary canal. The new bone formed at the end of the experiment was quantitated and related to the bioelectric potentials recorded at current-on and current-off states, respectively. Direct current stimulation resulted in electronegative potential spike centered on the cathode tip while current was applied. After electrical stimulation was turned off, the residual potentials at the end of the experiment did not significantly differ from the initial values. Conversely, the time sequence of the changes was significantly different from the control to the experimental group. The variations in the induced potentials at current-on state were significantly related to the variations in bone formation. This study suggests the existence of a relationship among bioelectric potentials, DC stimulation, and osteogenesis.

[Effect of insulin on the activity of bone alkaline phosphatase in culture]. / L'effetto dell'insulina sull'attività di fosfatasi alcalina dell'osso in coltura.

Diabetes and osteoporosis are linked. The question remains, however, as to whether insulin has any direct effect on bone formation. To test this hypothesis we have measured, as a marker of osteoblast activity, alkaline phosphatase (ALP) released by rat limb intact bones incubated in the presence and in the absence of physiological concentration of insulin. The results indicate that insulin significantly (p less than 0.012) increases ALP by a mean value of 48% (from 5.4% to 215%) over matched controls. We conclude that insulin has a direct stimulatory effect on osteoblast activity, and that in the absence of this effect, as in diabetes, bone loss might occur.

[The extracellular ionic environment and bone metabolism. In vitro study of the role of chloride]. / Ambiente ionico extracellulare e metabolismo osseo. Studio in vitro sul ruolo del cloro.

Controversies surround the view that the blood-bone ionic equilibrium is controlled by ionic changes at a so called "bone membrane" that display a pump-leak mechanism able to maintain a concentration gradient for chloride in addition to that for calcium, sodium and potassium. On the other hand it is known that a chloride-bicarbonate transport is active at bone cells membrane to control cytosolic pH. We tested the hypothesis that a modification of the chloride concentration of the culture medium, as well as a block of the chloride-bicarbonate transport by an anionic channels blocker, in an experimental model that keeps intact the bone membrane, may influence the bone cells activity and that the influence may be quantitatively different to that previously observed in isolated bone cells. The experimental model is the intact fetal rat limb bone, cultured for 24 hours in a simplified medium at different chloride concentrations obtained by substituting chloride with isethionate. Alkaline phosphatase activity released in the medium was measured as a marker of osteoblasts activity. By progressively reducing the chloride concentration from 117 mM to 29 mM, alkaline phosphatase activity was found unchanged. By dramatically reducing the medium chloride concentration to the range 14-0 mM, alkaline phosphatase activity was significantly (p = 0.0002) inhibited by 31.58%.(ABSTRACT TRUNCATED AT 250 WORDS)

Time-relationship between bone growth and increment of bone mineral content in growing rats.

The aim of the study was to describe the time-relationship between the growth rate of the rat tibia and the increase rate of its mineral content. Appositional and endochondral bone growth rates were derived from sequential X-rays measurements of tibial widening and lengthening, respectively; the increase rate of bone mineral content was derived from sequential photon absorptiometry measurements of the proximal tibio-fibular site. The time-relationship of appositional growth rate and endochondral growth rate versus bone mineral content increase rate was mathematically described. The results allow a better understanding of the time-course of two distinct features of bone growth: increase in size and increase in mineral content.

[The blood calcium error induced by oral calcium loading: study of the homeostatic compensation mechanism]. / L'errore calcemico indotto dal carico orale di calcio: studio dei meccanismi omeostatici di correzione.

The oral calcium load test, originally proposed for evaluating the intestinal calcium absorption and the renal calcium leak triggers some endocrine and metabolic responses addressed to correct the "calcemic error" induced by the load. Besides the increased plasma calcium there are: plasma PTH drop, increment in the urinary calcium excretion and in the threshold of tubular phosphate reabsorption. These responses have been measured and reciprocally correlated in 9 young adults at different times after the oral calcium load. The responses can be assessed with high precision in clinical practice and are in agreement with the known physiological models. The oral calcium load test is proposed as a tool for studying in the osteopenic population in the individual's capacity of correcting the calcemic error induced by the load.

[Reduction in parathormone secretion after oral calcium loading in osteoporotic adults]. / La riduzione della secrezione di paratormone dopo carico orale di calcio negli adulti osteoporotici.

The purpose of this study was to verify if a decreased inhibition of PTH secretion (abnormal suppressibility) in response to physiological increment of plasma calcium is present in patients with osteoporosis. The plasma concentration curve of intact PTH 1-84 following an oral calcium load (Pak) has been calculated in a selected population of 38 osteopenic patients (16 males and 22 females) and in a control group of 9 young healthy adults. All the patients included in this study a) had no past or present diseases and medications of potential influence on calcium homeostasis, b) showed a maximal calcemic response to the oral calcium load equal to that of the control group. PTH suppressibility was significantly smaller in the osteoporotic patients (-42% in males and -32% in females) than in the control group (-76%). This abnormal suppressibility of PTH is independent on sex and, in the females, also on postmenopausal estrogen deficiency. These results support the hypothesis that osteoporosis is associated to an altered secretory response of parathyroid glands maybe due to reduced sensitivity of the parathyroid cells to extracellular calcium.