Increase In Cancellous Bone Volume Research Articles

Effect of anodized zirconium implants on early osseointegration process in adult rats: a histological and histomorphometric study

Since surface plays a key role in bioactivity, the response of the host to the biomaterial will determine the success or failure of the prosthesis. The purpose of this study is to make an exhaustive analysis of the histological and histochemical characteristics of new bone tissue around Zr implants anodized at 60 V (Zr60) supported by histomorphometric methods in a rat model. Fibrous tissue was observed around the control implants (Zr0) and osteoblasts were identified on the trabeculae close to the implantation site that showed typical cytological characteristics of active secretory cells, regardless of the surface condition. The histomorphometrical analysis revealed a significant increase in cancellous bone volume, trabecular thickness and in trabecular number together with a decrease in trabecular separation facing Zr60. TRAP staining showed that there was a relative increase in the number of osteoclasts for Zr60. In addition, a larger number of osteoclast with a greater number of nuclei were detected in the tibiae for Zr60. This research demonstrated that the new bone microarchitecture in contact with Zr60 is able to improve the early stages of the osseointegration process and consequently the primary stability of implants which is a crucial factor to reduce recovery time for patients.

A Novel Bruton′s Tyrosine Kinase Inhibitor CC-292 In Combination With The Proteasome Inhibitor Carfilzomib Iimpacts Multiple Myeloma Bone Microenviroment With Resultant Anti-Myeloma Activity

A member of the Tec family kinases, Bruton's tyrosine kinase (Btk) modulates B-cell development and activation, and plays an important role in antibody production. Interestingly, Btk and Tec (the other Tec kinase family) regulate osteoclast (OC) differentiation via Receptor Activator of Nuclear Factor κ B (RANK) signaling. Moreover, OCs derived from X-linked agammaglobulinemia (XLA) patients who harbor Btk null mutations have impaired function.Here we show that a potent and specific Btk inhibitor, CC-292 inhibits OC function in multiple myeloma (MM) patients. CC-292 is a highly selective, covalent Btk inhibitor. OC derived from MM patient monocytes were assayed with or without CC-292. Interestingly, OC function was significantly inhibited in the presence of CC-292 (100 nM and 1000 nM) as demonstrated by pit formation assay. However, mRNA expression for TRAP and Cathepsin K, two OC differentiation markers were increased in the presence of CC-292 suggesting that CC-292 inhibits OC function without inhibiting OC differentiation.OC sealing zone contributes to OC bone resorption function. Given the role of c-Src and Proline-rich tyrosine Kinase 2 (Pyk2) signaling in sealing zone formation and OC function we next evaluated CC-292's effect on Pyk2 and c-Src. Pyk2 plays a role in OC activation and localizes to the sealing zone in OC. RANK signaling activates c-Src, which phosphorylates Pyk2. Moreover c-Src controls OC bone resorption by regulating actin organization via cortactin. Interestingly, CC-292 (100 nM) inhibited c-Src total protein, c-Src phosphorylation and Pyk2 phosphorylation. Furthermore, CC-292 (100 nM) inhibited cortactin protein and mRNA expression, and upregulated c-Cbl protein (E3 ubiquitin ligase for c-Src) expression in OC derived from MM patient monocytes with resultant inhibition of OC sealing zone formation. However, at the same low doses (100 nM) CC-292 did not show any direct in vitro effect against MM cell viability.Because carfilzomib, a proteasome inhibitor that binds irreversibly to its target, has potent anti-MM activity and also inhibits OC resorptive activity, we studied CC-292 in combination with carfilzomib. Our data suggests that carfilzomib (1.25 nM) has no impact on OC sealing zone formation but inhibits OC differentiation. CC-292 (100 nM) in combination with carfilzomib (1.25 nM) inhibited not only sealing zone formation but also OC differentiation, resulting in stronger suppression of OC function than carfilzomib alone.The combination of CC-292 (30mg/kg p.o. for 5 days per week for 6 weeks) and carfilzomib (3 mg/kg i.v. x 2 days per week for 4 weeks and 2 mg/kg i.v. x 2 days per week for 2 weeks) significantly inhibited tumor burden and myeloma cell numbers in a diffuse NOD-SCID MM model. The calvarial cells derived from these mice treated with CC-292 alone, carfilzomib alone or the combination showed higher osteocalcin mRNA (osteoblast differentiation marker) expression. A specific bone resorption marker, carboxy-terminal telopeptide collagen crosslinks (CTX) in mouse serum was significantly inhibited in CC-292 and CC-292 in combination with carfilzomib treatment groups in comparison with control mice. Furthermore, 3D microCT reconstructions showed increase in cancellous bone volume in lumbar vertebrae in mice treated with CC-292 or carfilzomib, while the combination treatment resulted in an increase in cancellous bone volume in an additive manner.These data demonstrate that the novel BTK inhibitor CC-292 inhibits OC function through inhibition of OC sealing zone formation. Moreover, CC-292 in combination with carfilzomib augments effects against the bone microenvironment with resultant anti-MM activity. Disclosures:Arastu-Kapur:Onyx Pharmaceuticals, Inc.: Employment. Evans:Celgene Avilomics Research: Employment, Equity Ownership. Singh:Celgene Avilomics Research: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals, Inc.: Employment. Westlin:Celgene Avilomics Research: Employment, Equity Ownership. Raje:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Amgen: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding.

Notch and the Skeleton

Notch receptors are transmembrane receptors that regulate cell fate decisions. There are four Notch receptors in mammals. Upon binding to members of the Delta and Jagged family of transmembrane proteins, Notch is cleaved and the Notch intracellular domain (NICD) is released. NICD then translocates to the nucleus, where it associates with the CBF-1, Suppressor of Hairless, and Lag-2 (CSL) and Mastermind-Like (MAML) proteins. This complex activates the transcription of Notch target genes, such as Hairy Enhancer of Split (Hes) and Hes-related with YRPF motif (Hey). Notch signaling is critical for the regulation of mesenchymal stem cell differentiation. Misexpression of Notch in skeletal tissue indicates a role as an inhibitor of skeletal development and postnatal bone formation. Overexpression of Notch inhibits endochondral bone formation and osteoblastic differentiation, causing severe osteopenia. Conditional inactivation of Notch in the skeleton causes an increase in cancellous bone volume and enhanced osteoblastic differentiation. Notch ligands are expressed in the hematopoietic stem cell niche and are critical for the regulation of hematopoietic stem cell self-renewal. Dysregulation of Notch signaling is the underlying cause of diseases affecting the skeletal tissue, including Alagille syndrome, spondylocostal dysostosis, and possibly, osteosarcoma.

Alfacalcidol-Stimulated Focal Bone Formation on the Cancellous Surface and Increased Bone Formation on the Periosteal Surface of the Lumbar Vertebrae of Adult Female Rats

To investigate the skeletal effects of alfacalcidol alone or in combination with exercise in intact adult female rats. Seventy-four 8.5-month-old rats were orally administered 0, 0.005, 0.025, 0.05 or 0.1 microg/kg of alfacalcidol for 12 weeks, alone or in combination with exercise. Cancellous bone histomorphometric measurements were performed on the second lumbar vertebra. At 0.05 and 0.1 microg/kg, alfacalcidol caused a significant increase in cancellous bone volume, accompanied by an increase in trabecular architecture. Percent eroded surface, bone resorption and formation were suppressed by alfacalcidol treatment. However, mineral apposition rate was significantly increased, indicating osteoblast activity was increased. A positive balance between bone formation and resorption was observed in the rats treated with the highest dose of alfacalcidol. Alfacalcidol induced a unique bone formation site ("bouton") on the cancellous surface. These boutons connected adjacent trabeculae and increased trabecular thickness. They exhibited both smooth and scalloped cement lines, suggesting that they were formed by minimodeling- and remodeling-based bone formation. Furthermore, alfacalcidol at 0.1 microg/kg increased periosteal bone formation of the lumbar transverse processes. Bipedal stance exercise alone did not have an effect on bone balance and bone turnover. There were no interactions between alfacalcidol and bipedal stance exercise except for a decrease in bone resorption. Alfacalcidol exhibited both anti-catabolic and anabolic effects on bone in intact female rats. The effect of combined treatment with alfacalcidol and bipedal stance exercise was no better than that of alfacalcidol alone.

The anabolic effect of PTH on bone is attenuated by simultaneous glucocorticoid treatment

Glucocorticoids (GC) are used for the treatment of a wide spectrum of diseases because of their potent anti-inflammatory and immunosuppressive effects, and they are serious and common causes of secondary osteoporosis. Administration of intermittent parathyroid hormone (PTH) may induce formation of new bone and may counteract the bone loss induced by GC treatment. Effects of simultaneous PTH and GC treatment were investigated on bone biomechanics, static and dynamic histomorphometry, and bone metabolism. Twenty-seven-month-old female rats were divided randomly into the following groups: baseline, vehicle, PTH, GC, and PTH + GC. PTH (1–34) 25 μg/kg and GC (methylprednisolone) 2.5 mg/kg were injected subcutaneously each day for a treatment period of 8 weeks. The rats were labeled with fluorochromes 3 times during the experiment. Bone sections were studied by fluorescence microscopy. The PTH injections resulted in a 5-fold increase in cancellous bone volume. At the proximal tibia, PTH induced a pronounced formation of new cancellous bone which originated from the endocortical bone surfaces and from thin trabeculae. Formation and modeling of connections between trabeculae were observed. Similar but less pronounced structural changes were seen in the PTH + GC group. The compressive strength of the cancellous bone was increased by 6-fold in the PTH group compared with the vehicle group. GC partially inhibited the increase in compressive strength induced by PTH. Concerning cortical bone, PTH induced a pronounced increase in the endocortical bone formation rate (BFR) and a smaller increase in periosteal BFR. The combination of PTH + GC resulted in a partial inhibition of the PTH-induced increase in bone formation. Serum-osteocalcin was increased by 65% in the PTH group and reduced by 39% in the GC group. The pronounced anabolic effect of PTH injections on the endocortical and trabecular bone surfaces and less pronounced anabolic effect on periosteal surfaces were partially inhibited, but not prevented, by simultaneous GC treatment in old rats. Both cortical and cancellous bone possessed full mechanical competence after treatment with PTH + GC.

Decreased bone anabolic effect of basic fibroblast growth factor at fatty marrow sites in ovariectomized rats

The purpose of the study was to compare the bone anabolic effects of basic fibroblast growth factor (bFGF) at hematopoietic (red) and fatty (yellow) marrow sites in ovariectomized (ovx) rats. Female Sprague Dawley rats were subjected to ovariectomy or sham surgery at 3 months of age and maintained untreated for 2 months after surgery. Three groups of ovx rats were then injected intravenously with bFGF for 14 days at a dose of 200 μg/kg body weight. One group of bFGF-treated OVX rats was killed at the end of the treatment period, whereas the other two groups were killed at 7 or 14 days after withdrawal of bFGF treatment. Another group of ovx rats and a group of sham-operated control rats were treated intravenously with vehicle alone for 14 days. The proximal tibia and first lumbar vertebra, bone sites with hematopoietic marrow, as well as the distal tibia and caudal vertebra, bone sites with primarily fatty marrow, were processed undecalcified for quantitative bone histomorphometry. At the hematopoietic marrow sites, bFGF treatment induced a marked accumulation of osteoid, which calcified during the withdrawal period to result in a significant increase in cancellous bone volume. Osteoblast and osteoid surfaces were increased by at least a factor of 10 at these sites in bFGF-treated ovx rats before declining rapidly during the withdrawal period. In contrast, osteoid volume was negligible in the fatty marrow sites of bFGF-treated ovx rats. Although these animals exhibited a nonsignificant trend for increased cancellous bone volume in the fatty distal tibia during the withdrawal period, no such trend was observed in the fatty caudal vertebra. Indices of bone formation (osteoblast and osteoid surfaces) were significantly increased by bFGF treatment in the fatty distal tibia, which retained some small pockets of hematopoietic cells, but not to the same great extent as in the skeletal sites with hematopoietic marrow. Furthermore, not even a trend for increased osteoblast and osteoid surfaces was observed in the fatty caudal vertebra of bFGF-treated ovx rats. These findings indicate that bFGF is a strong bone anabolic agent at skeletal sites with hematopoietic marrow, but the stimulatory effects of the growth factor on bone formation are greatly attenuated at fatty marrow sites.

Anabolic effect of estrogen replacement on bone in postmenopausal women with osteoporosis: histomorphometric evidence in a longitudinal study.

It is well recognized that estrogen (E(2)) prevents postmenopausal bone loss by suppressing bone resorption. Despite evidence that E(2) may also stimulate bone formation in animals, an anabolic effect in humans is still controversial. To investigate this, we studied 22 older postmenopausal females, with a mean age of 65.4 yr and mean interval of 16.9 yr since menopause and low bone mineral density. Transcortical iliac bone biopsies were performed before and 6 yr after E(2) replacement therapy (ERT) [75 mg percutaneous E(2) replaced 6-monthly plus oral medroxy progesterone acetate (5 mg daily) for 10 days each calendar month]. The mean serum E(2) level after 6 yr of treatment was 1077 (range, 180-2568) pmol/L. Bone mineral density improved in every patient, with a median increase of 31.4% at the lumbar spine and 15.1% at the proximal femur. Bone histomorphometry showed an increase in cancellous bone volume from 10.75% to 17.31% (P < 0.001). The wall thickness after 6 yr of E(2) treatment was 38.30 micrometer compared with 31.20 micrometer before commencement of ERT (P < 0.0005), indicating net bone gain. This is the first report showing histological evidence for an increase in cancellous bone volume, together with an increase in wall thickness, in a longitudinal follow-up study of ERT in older postmenopausal women. Our results show that E(2) is capable of exerting an anabolic effect in women with osteoporosis, even when started well into the menopause.

High-dose estrogen-induced osteogenesis in the mouse is partially suppressed by indomethacin

We recently found that high-dose estrogen induces the formation of new sites of cancellous bone formation within the long bones of intact female mice. To examine whether prostaglandins play a role in mediating this response, we studied whether this is inhibited by coadministration of the cyclooxygenase inhibitor, indomethacin. Eight-week-old intact female mice were divided into four groups of ten, and administered vehicle, 17β-estradiol (E 2), at 500 μg/animal per week and/or indomethacin at 2 mg/kg per day. Animals were killed after treatment for 24 days, and histomorphometric indices subsequently analyzed on longitudinal sections of the proximal tibial metaphysis. As found previously, E 2 treatment caused a striking increase in cancellous bone volume, associated with an equivalent increase in the extent of cancellous double-labeled surfaces. In mice treated with both indomethacin and E 2, significant reductions in cancellous bone volume and cancellous double-labeled surfaces were observed as compared with animals treated with E 2 alone. In contrast, indomethacin did not significantly influence these parameters when given alone. Subregional analysis within the proximal tibial metaphysis revealed that this inhibitory effect of indomethacin was more marked distally as compared with proximally, with the estrogen-induced gain in cancellous bone volume at these sites being reduced by 50% and 25%, respectively. We conclude that estrogen-induced osteogenesis in female mice is partially suppressed by treatment with indomethacin, suggesting that prostaglandin synthesis plays a significant role in mediating this response.

Growth hormone increases cortical and cancellous bone mass in young growing rats with glucocorticoid-induced osteopenia.

The effects of growth hormone (GH) on linear growth, bone formation, and bone mass have been examined in glucocorticoid (GC)-injected young growing rats. Two-month-old female Wistar rats were injected for 90 days with 1, 3, 6, or 9 mg of methylprednisolone alone or in combination with 5 mg of GH. Bone mass and bone formation parameters were examined in the femoral cortical bone and in cortical bone and cancellous bone of the lumbar vertebra. GC administration dose dependently decreased growth, longitudinal growth of the vertebra, as well as the modeling drift of the cortical bone of the vertebral body and femoral diaphysis. In the vertebral cancellous bone, GC also decreased the mineralizing surface and inhibited the growth-related increase in cancellous bone volume. GH increased growth, longitudinal growth of the vertebra, as well as the modeling drift of the vertebral body and the femoral diaphysis, resulting in an increased cortical bone mass. GH also increased cancellous bone volume and the mineralizing surface of the vertebral body. In GC-injected animals, GH normalized and further increased growth, longitudinal growth, and the modeling drift of both the femoral diaphysis and the vertebral body, resulting in an increased cortical bone mass at both locations. GH also increased cancellous bone volume of the vertebral body in GC-injected animals, but GH did not, however, reverse the decreased mineralizing surface of cancellous bone induced by GC injections. In conclusion, GC administration to growing rats retards normal growth, longitudinal growth, and cortical bone modeling drift. It also decreases the cancellous bone mineralizing surface and inhibits the normal age-related increase in cancellous bone volume of the vertebral body. In the growing rat skeleton, GH can counteract these GC-induced side effects, except for the GC-induced decrease in the mineralizing surface of cancellous bone of the vertebral body, which remained unaffected by GH administration.

The impact of mammalian reproduction on cancellous bone architecture.

Pregnancy and lactation make demands on maternal calcium homeostasis which may affect bone strength. Recently, changes in cancellous architecture have been described in iliac crest bone biopsies from normal pregnant women but the rarity of such human material means an animal model is essential. The microanatomy of cancellous bone was compared in uniparous and multiparous rats using undecalcified histological sections of lumbar and caudal vertebrae and also proximal femora. An automated trabecular analysis system (TAS) measured a comprehensive range of structural variables including the trabecular number, connectivity and width. In the first pregnancy cycle an early stimulation of bone formation (which quadrupled at some sites) was indicated by an increase in the skeletal uptake and spacing of double calcein labels and the immediate generation of thicker more numerous and interconnected trabeculae. A 40% increase in cancellous bone volume was observed in the lumbar spine in comparison with age-matched virgin controls. In contrast, a rapid succession of 3 pregnancy cycles (including lactation) culminated in cancellous atrophy of 15% at the same site, with a loss in trabecular number ranging from 20% (caudal vertebra) to 30% (lumbar vertebrae). In comparison, the proximal femur lost 40% of its struts but, nevertheless, uniquely sustained its cancellous bone volume. When lactation was excluded the number of struts lost was halved although trabecular thinning then took place which was sufficient to maintain the previous 15% deficit in bone volume. It was concluded that a single pregnancy strengthens the cancellous component of the maternal skeleton while a quick succession of pregnancies weakens it. Lactation influences the pattern of bone loss but not its amount.

Differential effect of treadmill exercise on three cancellous bone sites in the young growing rat

The aim of the present study was to examine cancellous bone changes induced by exercise on three different skeletal sites, the lumbar vertebra, the proximal, and the distal tibia, in the young growing rat. Forty 4-week-old female Sprague-Dawley rats were randomized into 4 groups of 10 animals each; 8 weeks exercise (8EX), 8 weeks sedentary control (8CON), 12 weeks exercise (12EX), and 12 weeks sedentary control (12CON). The exercise regimen consisted of treadmill running at 24 m/min 1 hr per day 5 days a week. After each period of exercise, the proximal and distal tibial metaphyses (PTM and DTM, respectively) and the fifth lumbar (L5) vertebral body were processed for histomorphometry of the cancellous bone (secondary spongiosa) and cortical periosteum. Eight and twelve weeks of exercise significantly increased the mineral apposition rate and bone formation rate in the PTM and DTM, and 12 weeks of exercise significantly increased the labeled perimeter in the DTM, compared with the age-matched controls. Eight and twelve weeks of exercise significantly increased cancellous bone volume in the PTM (mean ± standard deviation, 8EX; 19.1 ± 2.9% vs 8CON; 14.3 ± 3.1%, P < 0.05 and 12EX; 18.8 ± 3.5% vs 12CON; 15.2 ± 3.3%, P < 0.05), and 12 weeks exercise significantly increased cancellous bone volume in the DTM, compared with age-matched control (12EX; 32.5 ± 7.7%, 12CON; 22.2 ± 4.8%, P < 0.05). The increase in cancellous bone volume by 12 weeks exercise was higher in the DTM than that in the PTM (43.4% and 24.0%, respectively). On the other hand, the exercise did not significantly affect cancellous bone volume and bone formation in the L5 vertebral body, although the cortical periosteal bone formation rate and the L5 vertebral bone mass were increased. These findings suggest that cancellous bone adaptation to treadmill exercise is site specific, and the effect may be influenced by factors such as mechanical loading and metaphyseal bone architecture in the young growing rat.

Administration of a Glucocorticoid With Depot Effect Counteracts the Stimulating Effect of Growth Hormone on Cancellous and Cortical Bone of the Vertebral Body in Rats

Our earlier studies have shown that growth hormone administration could not counteract decreased longitudinal growth and cortical osteopenia of rat femora induced by a glucocorticoid with depot effect. In the present study we examined the effects of glucocorticoid on vertebral bone as well as the effect of growth hormone on vertebral bone in young growing animals also given glucocorticoid injections. Five groups of female rats (3 1/2 months) were treated for 80 days as follows: (1) saline, (2) prednisolone: Delcortol 5 mg/kg/day, (3) growth hormone: 5 mg/kg/day, (4) prednisolone and growth hormone, (5) food restriction. Vertebral dimensions, histomorphometry, and mechanical competence of the vertebral bone were examined. Growth hormone administration increased body weight, vertebral height, cross-sectional area, and volume. The compressive strength of the L4-corpus cylinder was also increased due to an increase in cancellous bone volume and an increase in the area of cortical bone surrounding the vertebral body. Glucocorticoid administration decreased body weight, height, and volume of the intact vertebrae. Histological examination revealed that glucocorticoid administration decreased the area of cortical bone surrounding the vertebral body but had no effect on the cancellous bone volume. No effect of glucocorticoid administration on mechanical strength of the L4 corpus cylinder could be detected. In agreement with our findings in cortical bone, we found no effect of growth hormone on vertebral bone when given to animals also receiving glucocorticoid injections. Growth hormone increases longitudinal growth, cortical and cancellous bone mass, and mechanical competence of the vertebral body. Glucocorticoid administration decreases longitudinal growth of the vertebrae and cortical bone mass without affecting cancellous bone mass of the vertebral body. Despite this, administration of a glucocorticoid with depot effect totally inhibits the effect of growth hormone on vertebral bone.

Effects of moderate intensity exercise on tibial bone mass in mature ovariectomized rats: bone histomorphometry study.

The effects of moderate intensity exercise of different duration on the cancellous and cortical bone mass of the tibia were investigated in mature ovariectomized rats by bone histomorphometry. A total of twenty 23-week-old female Wistar rats were used in this study. Ovariectomy (OV) was performed on 15 animals, and they were divided into three groups of five animals each: an OV group, an OV + exercise for 30 min a day (EX30) group and an OV + EX60 group. The other 5 animals served as a sham-operated (SH) control group. The exercise consisted of treadmill running at 16 m/min and was performed 5 days a week for 12 weeks after the operation. After 12 weeks of exercise, bone histomorphometry was used to evaluate the cancellous bone (secondary spongiosa) of the proximal tibia and the cortical bone of the tibial shaft. The OV group showed a significant decrease in cancellous bone volume, with increased resorption and formation, compared to the SH group (P < 0.01). Although the OV + EX30 group showed a significant increase in cancellous bone volume, with decreased resorption and increased osteoblastic activity, compared to the OV group (P < 0.05), cancellous bone volume in the OV + EX30 group remained significantly lower than that in the SH group (P < 0.05). There was not a significant difference in cortical bone area between the OV group and the SH group, whereas the OV + EX30 group showed a significant increase in cortical bone area, with decreased resorption on the endosteal surface, compared to the OV group (P < 0.05). There were no significant differences in cancellous bone volume or cortical bone area between the OV group and the OV + EX60 group. The data suggest that moderate intensity exercise of appropriate duration may attenuate ovariectomy-induced cancellous bone loss and increase cortical bone mass in mature ovariectomized rats.

Distinct effects of ovarian transplantation and exogenous 17 β-oestradiol on cancellous bone of osteopenic ovariectomized rats

Although 17 beta-oestradiol (E2) is known to prevent bone loss, prolonged administration of E2 is unable to reverse this in female rats rendered osteopenic by ovariectomy. To determine whether this reflects a failure to replace other components of ovarian function involved in bone metabolism, we compared the effects of administering E2 to osteopenic ovariectomized (ovx) rats with those of ovarian transplantation. Ovariectomy was performed in female rats. After 13 weeks, by which time marked bone loss had occurred, one group of ovx animals received ovaries from donor rats, and, after a delay of 2 weeks to allow oestrus cycles to return, a further group received E2 5 micrograms.kg-1.day-1 for 9 weeks. The dose of E2 was chosen as that which in preliminary studies restored mean serum E2 levels to that of intact female rats. The study was terminated 24 weeks after ovariectomy. Both E2 and ovarian transplantation largely restored indices of oestrogenic exposure in ovx rats to those of sham-ovx animals. Animals receiving ovarian transplants also showed a small increase in serum progesterone and full restoration of serum testosterone. However, while ovarian transplantation also returned indices of cancellous bone metabolism to those of sham-ovx animals, there was little increase in bone volume. Interestingly, exogenous E2 caused a greater increase in cancellous bone volume than ovarian transplantation but also caused more marked suppression of bone formation, as assessed at the end of the study. In conclusion, exogenous E2 and ovarian transplantation exerted distinct effects on skeletal metabolism in osteopenic ovx rats, although the basis for this difference is currently unclear.

A histomorphometric study of bone changes in thyroid dysfunction in rats

Clinical studies in thyrotoxicosis reveal a state of high bone turnover leading, eventually, to osteoporosis. Recently there has been concern that thyroxine (T4) treatment may have a similar effect on bone. Rat models have been used to study the effects of T4 on bone, but the majority of studies have looked at the effects of T4 after only 3 weeks of treatment. The aim of this study was to evaluate histomorphometric changes in rats after 12 weeks of thyroxine overtreatment or 12 weeks of hypothyroidism compared with untreated control animals. Animals received either T4 200 micrograms/kg per day, 0.1% propylthiouracil, or vehicle for 12 weeks. Tetracycline was administered 1 week and 3 weeks prior to killing. Iliac crest bone was used for histomorphometry. Serum T4 measurements (taken at killing) confirmed hyper- and hypothyroidism in the appropriate animal groups (between group difference p < 0.001 by ANOVA). In hyperthyroid animals there was an increase in mineral apposition rate (MAR; 0.94 vs. 0.59 microns/day, p < 0.001) and mineral formation rate (MFR/BS; 0.24 vs. 0.12 x 10(-2) micron3/micron2 per day, p < 0.001) and a slight increase in eroded surfaces (ES/BS%; 1.54 vs. 1.36, p < 0.05) compared with controls, consistent with previous in vitro and in vivo observations. In hypothyroid rats there was a marked reduction in osteoid surfaces (OS/BS%; 1.7 vs. 24.8, p < 0.001) and MAR (0.3 vs. 0.59 micrograms/day, p < 0.001), a reduction in ES/BS% (0.51 vs. 1.36, p < 0.05), and an increase in cancellous bone volume (BV/TV%; 30.29 vs. 19.6, p < 0.05), suggesting that thyroid hormones are a requirement for normal bone turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclical treatment of osteopenic ovariectomized adult rats with PTH(1–34) and pamidronate

Administration of PTH(1-34) at low intermittent doses stimulates bone formation in vivo. However, the new bone is quickly resorbed after drug withdrawal. Following the concept of lose, restore, and maintain, we studied the possibility of maintaining the PTH(1-34)-mediated new bone by APD post-treatment and the effect of a second PTH(1-34)-APD cycle in osteopenic ovariectomized (OVX) adult rats. Eighty OVX rats (6-months-old, 3 months after OVX) and 10 sham-operated rats were divided into nine groups. One OVX group was killed as baseline (BL). Five OVX groups were injected with PTH(1-34) (20 micrograms/kg/day, subcutaneously) for 3 weeks (5 days/week). The remaining two OVX groups and the sham group (S) were injected with saline as controls. One PTH(1-34) group (A) and one control group (B) were then killed. Three PTH(1-34) groups were post-treated with APD injections (250 micrograms/kg/day, subcutaneously) for 5 days; the remaining PTH(1-34) group, the other control group, and group S were injected with saline; and all these groups were left untreated for 3 weeks. At the end, one PTH-APD group (C), the PTH-saline group (D), and the saline-saline group (E) were killed. Another PTH-APD cycle was applied to the remaining two PTH-APD groups, with group F killed after the second PTH treatment and group G killed after two complete cycles. We found a 90% increase in cancellous bone volume (Cn.BV/TV) and a 28% increase in trabecular thickness (Tr.Th) in group A over group B.(ABSTRACT TRUNCATED AT 250 WORDS)

Intermittent retinoie acid in combination with continuous oestradiol-17β increases cancellous bone volume in osteopaenic ovariectomized rats

Although short-term administration of oestradiol-17 beta (OE2) stimulates cancellous bone formation in the rat, this is replaced by a tendency to suppression after prolonged treatment. Hence, in rats rendered osteopaenic by ovariectomy, OE2 administration fails either to induce a sustained increase in bone formation or to restore bone volume. A possible explanation for this failure is that OE2 also inhibits bone resorption, secondarily suppressing bone formation through coupling mechanisms. We therefore investigated whether the effects of OE2 treatment might be modified by intermittently stimulating bone resorption with retinoic acid (120mg/kg daily) for 4 out of every 20 days. We found, in a preliminary experiment using intact animals, that intermittent retinoic acid reduced cancellous bone volume, consistent with previously documented stimulation of bone resorption by retinoic acid. Rats were then rendered osteopaenic by ovariectomy, and given vehicle, retinoic acid and/or OE2. We found that animals treated with intermittent retinoic acid and OE2 showed a substantial increase in cancellous bone volume compared with ovariectomized animals treated with vehicle, retinoic acid alone or OE2 alone. Therefore, intermittent retinoic acid appears to cause a net increase in bone formation over resorption when given to ovariectomized animals in conjunction with OE2. We conclude that the effects of OE2 on cancellous bone are modified by intermittent treatment with retinoic acid, resulting in a substantial increase in bone volume.

Maintenance of cancellous bone connectivity in primary hyperparathyroidism: trabecular strut analysis.

We previously demonstrated an increase in cancellous bone volume and trabecular number in patients with mild primary hyperparathyroidism (PHPT). To test the hypothesis that this increase is due to preservation of cancellous bone architecture, we conducted a trabecular strut analysis using a new method that measures trabecular connectivity. Iliac crest biopsies from 37 patients with PHPT, 14 men (28-68 years) and 23 women (26-68 years), were examined histomorphometrically and compared to cadaveric samples from 24 age-matched subjects, 17 men and 7 women. Two-dimensional indices of cancellous structure--node number (N.Nd), terminus number (N.Tm), node to node (Nd.Nd), node to terminus (Nd.Tm), and terminus to terminus (Tm.Tm) strut lengths, and total strut length (TSL)--were measured and the ratio of node number to terminus number (N.Nd/N.Tm) calculated. TSL, N.Nd, and Nd.Nd were significantly higher in patients than in controls. TSL and Nd.Nd, but not N.Nd or Nd/Tm, decreased significantly with age in PHPT, indicating that age-related bone loss in PHPT occurs without significant loss of trabecular connectivity. Two-dimensional indices reflecting connectivity or the amount of bone, that is, N.Nd, Nd.Nd, N.Nd/N.Tm, and TSL, correlated positively with cancellous bone volume (BV/TV) and trabecular number (Tb.N) and negatively with trabecular spacing (Tb.Sp) in both PHPT and controls. Trabecular thickness (Tb.Th) correlated positively with Nd.Nd and Tb.N and negatively with Tm.Tm in PHPT but not in controls. The present data show that in PHPT there is not only greater cancellous bone volume and trabecular number but preserved trabecular connectivity as well. The data further support the hypothesis that in PHPT cancellous bone architecture is maintained.

Skeletal fluorosis: Histomorphometric analysis of bone changes and bone fluoride content in 29 patients

Bone fluoride content (BFC) was measured and histomorphometric analysis of undecalcified sections was performed in transiliac biopsy cores from 29 patients (16 men, 13 women, aged 51 ± 17 years) suffering from skeletal fluorosis due to chronic exposure to fluoride. The origin of the exposure, known in 20 patients, was either hydric (endemic or sporadic) or industrial, or in a few cases iatrogenic. Measured on calcined bone using a specific ion electrode, BFC was significantly high in each specimen ( mean ± SD; 0.79 ± 0.36% on bone ash). The radiologically evident osteosclerosis observed in each patient was confirmed by a significant increase in cancellous bone volume (40.1 ± 11.2% vs. 19.0 ± 2.8% in controls, p < 0.0001). There were significant increases in cortical width (1292 ± 395 mem vs. 934 ± 173 mcm, p < 0.0001) and porosity (14.4 ± 6.4% vs. 6.5 ± 1.7%, p < 0.002), but without reduction of cortical bone mass. Cancellous osteoid volume and perimeter, as well as width of osteoid seams, were significantly increased in fluorotic patients. The increase in cancellous osteoid perimeter was almost three-fold greater than that noted in cancellous eroded perimeter. In 15 patients doubly labeled with tetracycline, the mineral apposition rate was significantly decreased, mineralization lag time was significantly increased. The fluorotic group had a greater number of osteoblasts than controls with a very high proportion of flat osteoblasts. The ultrastructural characteristics reflecting the activity of the bone cells were clearly visible on electron microscopy. Bone formation rate and adjusted apposition rate were significantly decreased in skeletal fluorosis. On stained sections and microradiographs, bone tissue showed typical modifications for skeletal fluorosis (linear formation defects, mottled bone). The volume of cancellous interstitial mineralization defects and the proportion of mottled periosteocytic lacunae were markedly increased in skeletal fluorosis. These two parameters were significantly correlated together but neither of these was significantly correlated with BFC. Renal function did not significantly influence the changes in BFC and histomorphometry of fluorotic patients. Skeletal fluorosis is thus characterized by an unbalanced coupling in favor of bone formation, and a great number of osteoblasts with a high proportion of flat osteoblasts. This may explain the mineralization impairment proven by thick osteoid seams and reduced mineral apposition rate, and supports the view that fluoride may have a dual effect on osteoblasts: a probable increased birthrate at the tissue-level due to a mitogenic effect of fluoride on precursors of osteoblasts, and a toxic effect at the individual cell-level. The addition of these two effects represents, however, a marked increase of bone formation at the organ level.