Dynamic Histomorphometry Research Articles

Biochemical and histological assessment of Alkali therapy during high animal protein intake in the rat

The Westernized diet is acidogenic due to the high content of sulfur-containing amino acids and relative deficiency of potassium organic anions. Chronic acid loads result in hypercalciuria and negative calcium balance often associated with loss of bone mineral. Alkali therapy tends to reverse the hypercalciuria but little is known regarding its effect on bone as assessed by bone histomorphometry. The present study utilized dynamic bone histomorphometry to evaluate the effects of alkali therapy on acid-induced changes in bone turnover. Serum and urine analyses and bone histomorphometry were assessed in adult rats after 2 months of either a low casein (LC) or high casein (HC) diet supplemented with either potassium chloride (KCl) or potassium citrate (KCit). Compared to animals on LC-KCl diet, HC-KCl diet delivered a substantial acid load as shown by significant increases in urinary sulfate, ammonium, and net acid excretion, and a lower urinary pH and citrate excretion without detectable changes in serum parameters. The acid load also resulted in hypercalciuria. Dynamic and static bone histomorphometry disclosed a significant reduction in cancellous bone volume and trabecular number associated with a 2.5-fold increase in eroded and a 3.5-fold increase in osteoclastic surfaces. There was also a near 2-fold increase in bone formation rate in rats on the HC-KCl diet. When animals on the HC diet were given KCit instead of KCl, all of the aforementioned changes in urine biochemistry and bone turnover were significantly attenuated or entirely prevented. These findings underscore the deleterious effects of high animal protein intake in promoting hypercalciuria and increasing bone turnover. Co-administration of potassium alkali attenuates or prevents these changes. In this animal model of high dietary animal protein intake, the major skeletal effect of alkali therapy is to reduce bone resorption, with little or no effect on bone formation.

Epimedium-derived flavonoids promote osteoblastogenesis and suppress adipogenesis in bone marrow stromal cells while exerting an anabolic effect on osteoporotic bone

Background Epimedium-derived flavonoids (EFs) have been reported to prevent bone loss in ovariectomized (OVX) rats and late postmenopausal women but the underlying mechanism of the anabolic effect is unknown. Objective This study aimed to investigate the effect of EFs on osteoporotic bone using histomorphometry and on osteoblastogenesis/adipogenesis of bone marrow stromal cells (BMSCs). Methods 11-month-old female Wistar rats were divided into Sham, OVX, Sham + soluble vehicle (Sham + SV), OVX + SV and OVX + EFs (10 mg/kg/day) groups. 3 months after surgery, rats from the first two groups were euthanized to verify the establishment of OVX-induced osteoporosis. Other groups were orally treated with either daily SV or EFs for 4 months. At sacrifice, serum was analyzed for the levels of osteocalcin and TRACP 5b, BMD in the proximal femur was measured by pQCT. Static and dynamic bone histomorphometry was performed in proximal tibiae with microCT and undecalcified sections, respectively. The effect of EF treatment on differentiation of rat BMSCs was assessed by colony formation assays and gene expression analysis, respectively. Gene expression, ALP activity and adipocyte numbers were determined in differentiating human BMSCs after exposure to conditioned serum from SV- or EFs-treated OVX rats. Results The serum level of osteocalcin was higher and TRACP 5b was lower in EFs versus SV-treated OVX rats. BMD, BV/TV, Tb.N and Conn.D in EFs-treated OVX rats were significantly greater than those of SV-treated OVX rats. Bone histomorphometric parameters OS/BS, MAR, and BFR/BS were significantly higher in EFs versus SV-treated OVX rats. EFs significantly increased osteogenesis and decreased adipogenesis of BMSCs, as evidenced by CFU-ALP and CFU-Adipo assays, respectively. The mRNA level of Runx2 and bone sailoprotein was significantly higher while PPARγ2 was significantly lower in BMSCs from EFs-treated versus SV-treated OVX rats. ALP activity and Runx2 mRNA was significantly higher while adipocyte number and PPARγ2 mRNA was significantly lower in human BMSCs after exposure to conditioned serum from EFs versus SV-treated OVX rats. Conclusion EFs exerted anabolic effect on osteoporotic bone by concomitantly promoting osteogenic and suppressing adipogenic differentiation of BMSCs.

Mecp2 deficiency decreases bone formation and reduces bone volume in a rodent model of Rett syndrome

Rett syndrome (RTT), a neurological disorder characterized by neurological impairment and a high frequency of osteopenia which often manifests early in childhood, most often is caused by inactivating mutations in the X-linked gene encoding a regulator of epigenetic gene expression, methyl CpG binding protein, MeCP2. Clinical data show that, along with neurological defects, females with RTT frequently have marked decreases in bone mineral density (BMD) beyond that expected from disuse atrophy. To investigate the relationship between loss of Mecp2 and reduced BMD, we used a Mecp2 null mouse model, Mecp2 −/yBIRD, for our histological and biochemical studies. Mecp2 −/yBIRD mice have significantly shorter femurs and an overall reduced skeletal size compared to wild-type mice by post-natal day 60 (P60). Histological and histomorphometric studies identified growth plate abnormalities as well as decreased cortical and trabecular bone in P21 and especially in P60 Mecp2 −/yBIRD mice. Dynamic histomorphometry revealed decreased mineral apposition rates (MAR) in Mecp2 null femoral trabecular bone as well as in calvarial bone samples. While changes in MAR of cortical bone were not significant, loss of Mecp2 significantly reduced cortical, trabecular and calvarial bone volume compared with age-matched wild-type animals. These differences indicate that Mecp2 deficiency leads to osteoblast dysfunction, which translates into reduced osteoid deposition accounting for the reduced bone volume phenotype. While individual variations were observed in OPG and Rankl concentrations, molar ratios of OPG:Rankl at P21 and P60 were comparable between wild-type and Mecp2 −/yBIRD mice and showed a consistent excess of OPG. In tibial sections, TRAP staining demonstrated equivalent osteoclast number per bone surface measurements between wild-type and null animals. Our work with a Mecp2 null mouse model suggests epigenetic regulation of bone in the Mecp2 −/yBIRD mice which is associated with decreased osteoblast activity rather than increased osteoclastic bone loss.

New Roles for INSL3 in Adults

Insulin-like factor 3 (INSL3) is produced primarily by testicular Leydig cells and acts by binding to its specific G-protein-coupled receptor, RXFP2 (relaxin family peptide 2). INSL3 is involved in testicular descent during fetal development, and mutations in the INSL3 and RXFP2 genes cause cryptorchidism. The physiological role of INSL3 in adults is not known, although substantial INSL3 circulating levels are present. After extensive clinical, biochemical, and hormonal investigations, including bone densitometry with dual energy X-ray absorptiometry, on 25 young men (age, 27-41 years) who have the well-characterized T222P mutation in the RXFP2 gene, we found that 16 of them (64%) had significantly reduced bone density. No other cause of osteoporosis was evident in these subjects, whose testosterone and gonadal function were normal. Expression analysis of INSL3 and RXFP2 on human bone biopsy and human and mouse osteoblast cell cultures performed by reverse transcription-PCR and immunohistochemistry showed the presence of RXFP2 in these cells. Real-time cAMP imaging analysis and proliferation assays under the stimulus of INSL3 showed a dose- and time-dependent increase in cAMP and cell proliferation, and specific osteoblast gene activation was observed by real-time PCR after INSL3 stimulation. Lumbar spine and femoral bone of Rxfp2-deficient mice were studied by static and dynamic histomorphometry and micro-computed tomography, respectively, and showed decreased bone mass, mineralizing surface, bone formation, and osteoclast surface compared to wild-type littermates, compatible with a functional osteoblast impairment. This study identified for the first time a role for INSL3 in adults, demonstrating a modulating effect on bone metabolism and linking RXFP2 gene mutations with human osteoporosis.

Interleukin-1 is essential for systemic inflammatory bone loss

Objectives:Chronic inflammation is a major risk factor for systemic bone loss leading to osteoporotic fracture and substantial morbidity and mortality. Inflammatory cytokines, particularly tumour necrosis factor (TNF) and interleukin-1 (IL1),...

Dextran sodium sulfate-induced colitis causes rapid bone loss in mice

Introduction Osteopenia is a common complication of human inflammatory bowel disease (IBD). We evaluated the contribution of colonic inflammation to osteopenia and its mechanism in a murine colitis model. Methods Colitis was induced by adding dextran sodium sulfate (DSS) to the drinking water for 2 weeks to nine-week-old Balb/C male mice. 5% DSS was added on the first week and was reduced to 2.5% on the second week. Age- and sex-matched Balb/C mice served as the control group. Indices of femoral bone mass and architecture were determined by micro computed tomography (μCT). Bone formation parameters and osteoclast number were determined by dynamic histomorphometry. The degree of colonic inflammation was assessed by a clinical disease activity index, and colonic mucosal myeloperoxidase activity. Results DSS-treated mice exhibited a significantly lower bone mass compared to controls as indicated by decreased trabecular bone volume (BV/TV) of 32%. This reduction was accompanied by decreased trabecular number (23%) and connectivity density (37%) compared to the controls. No changes were observed in cortical bone indices. Osteopenia resulted from suppressed bone formation, as indicated by decreased trabecular double-labeled surface (dL%) of 90%, mineralizing surface (MS) of 62%, and bone formation rate (BFR) of 67%, and increased bone resorption as indicated by a 34% increase in osteoclast number in DSS-treated mice compared to the controls. Myeloperoxidase activity inversely correlated with trabecular BV/TV ( r = − 0.67, p = 0.02), trabecular number ( r = − 0.86, p = 0.0008) and connectivity density ( r = − 0.63, p = 0.03). Myeloperoxidase activity inversely correlated with the bone formation indices: dL%, MS, and BFR ( r = − 0.79, p = 0.007, r = − 0.84, p = 0.002, r = − 0.83, p = 0.003, respectively). Conclusions DSS-induced colitis is associated with reduced femoral bone mass and altered micro architecture, which results from suppressed bone formation and increased bone resorption. The decrease in indices of bone mass, structure and formation are directly linked to the degree of colonic mucosal inflammation. DSS-induced colitis can be used to study pharmacological interventions for bone loss in colitis.

Effects of genistein on vertebral trabecular bone microstructure, bone mineral density, microcracks, osteocyte density, and bone strength in ovariectomized rats

Until now, the effects of phytoestrogen on bone in both women and ovarian hormone-deficient animal models of osteoporosis have remained uncertain. We have aimed here to investigate the effect of genistein (GEN) on trabecular bone quality in ovariectomized (OVX) rats. Forty 7-month-old female Sprague-Dawley rats were randomly divided into the following four groups: OVX, sham-operated (SHAM), treated with 17beta-estradiol (EST, 10 microg x kg(-1) x day(-1)), and GEN (5 mg x kg(-1) x day(-1)). At 15 weeks postoperation, the compressive test was performed on the L5 vertebral body; additionally, microcomputed tomography (micro-CT) assessment was performed to estimate the bone mineral density (BMD) and microstructure parameters of the L6 vertebral body. After fatigue damage testing, the L6 vertebral body was bulk-stained in 1% basic fuchsin and embedded in methylmethacrylate. The L4 vertebral body was embedded in methylmethacrylate for dynamic histomorphometry analysis without staining. Mounted bone slices were used to measure microcrack parameters, empty osteocyte lacuna density (e.Lc.Dn), and osteocyte density (Ot.N/T.Ar). Maximum loading (ML) and Ot.N/T.Ar were significantly lower in the OVX group than in the other groups. E.Lc.Dn was significantly decreased in GEN and EST groups compared to the OVX group. ML was significantly decreased in the GEN group compared to the SHAM group. Microcrack density, microcrack surface density, and microcrack length were significantly increased in the OVX group compared to the other groups. Mineral apposition rate was significantly decreased in the OVX group compared to the SHAM and GEN groups. Bone formation rate was significantly decreased in the OVX group compared to other groups. There were no significant differences with regard to mineralizing surface among the four groups. Volumetric BMD at organ was significantly lower in OVX, EST, and GEN groups than in the SHAM group. Bone mineral content was significantly lower in the OVX group than in the SHAM group. Bone volume fraction and trabecular number were significantly decreased in OVX, EST, and GEN groups compared to the SHAM group. Structure model index was significantly lower in the SHAM group than in OVX, EST, and GEN groups. Trabecular separation was significantly increased in the OVX group compared to SHAM and EST groups. There were no significant differences with regard to the trabecular thickness (Tb,Th) between SHAM, GEN, and OVX groups. Tb.Th was significantly lower in the EST group than in the SHAM group. Connectivity density (Conn.D) was significantly lower in the OVX group than in SHAM and GEN groups, and Conn. D was significantly lower in the EST group than in GEN. In conclusion, the present study demonstrates that GEN preserved the biomechanical quality of the trabecular bone regardless of the microstructure and BMD.

Bone Mass Is Preserved and Cancellous Architecture Altered Due to Cyclic Loading of the Mouse Tibia After Orchidectomy

The study of adaptation to mechanical loading under osteopenic conditions is relevant to the development of osteoporotic fracture prevention strategies. We previously showed that loading increased cancellous bone volume fraction and trabecular thickness in normal male mice. In this study, we tested the hypothesis that cyclic mechanical loading of the mouse tibia inhibits orchidectomy (ORX)-associated cancellous bone loss. Ten-week-old male C57BL/6 mice had in vivo cyclic axial compressive loads applied to one tibia every day, 5 d/wk, for 6 wk after ORX or sham operation. Adaptation of proximal cancellous and diaphyseal cortical bone was characterized by muCT and dynamic histomorphometry. Comparisons were made between loaded and nonloaded contralateral limbs and between the limbs of ORX (n = 10), sham (n = 11), and basal (n = 12) groups and tested by two-factor ANOVA with interaction. Cyclic loading inhibited bone loss after ORX, maintaining absolute bone mass at age-matched sham levels. Relative to sham, ORX resulted in significant loss of cancellous bone volume fraction (-78%) and trabecular number (-35%), increased trabecular separation (67%), no change in trabecular thickness, and smaller loss of diaphyseal cortical properties, consistent with other studies. Proximal cancellous bone volume fraction was greater with loading (ORX: 290%, sham: 68%) than in contralateral nonloaded tibias. Furthermore, trabeculae thickened with loading (ORX: 108%, sham: 48%). Dynamic cancellous bone histomorphometry indicated that loading was associated with greater mineral apposition rates (ORX: 32%, sham: 12%) and smaller percent mineralizing surfaces (ORX: -47%, sham: -39%) in the final week. Loading resulted in greater BMC (ORX: 21%, sham: 15%) and maximum moment of inertia (ORX: 39%, sham: 24%) at the cortical midshaft. This study shows that cancellous bone mass loss can be prevented by mechanical loading after hormonal compromise and supports further exploration of nonpharmacologic measures to prevent rapid-onset osteopenia and associated fractures.

Mutations in the Insulin-Like Factor 3 Receptor Are Associated With Osteoporosis

Insulin-like factor 3 (INSL3) is produced primarily by testicular Leydig cells. It acts by binding to its specific G protein-coupled receptor RXFP2 (relaxin family peptide 2) and is involved in testicular descent during fetal development. The physiological role of INSL3 in adults is not known, although substantial INSL3 circulating levels are present. The aim of this study was to verify whether reduced INSL3 activity could cause or contribute to some signs of hypogonadism, such as reduced BMD, currently attributed to testosterone deficiency. Extensive clinical, biochemical, and hormonal study, including bone densitometry by DXA, was performed on 25 young men (age, 27-41 yr) with the well-characterized T222P mutation in the RXFP2 gene. Expression analysis of INSL3 and RXFP2 on human bone biopsy and human and mouse osteoblast cell cultures was performed by RT-PCR, quantitative RT-PCR, and immunohistochemistry. Real-time cAMP imaging analysis and proliferation assay under the stimulus of INSL3 was performed on these cells. Lumbar spine and femoral bone of Rxfp2-deficient mice were studied by static and dynamic histomorphometry and muCT, respectively. Sixteen of 25 (64%) young men with RXFP2 mutations had significantly reduced BMD. No other apparent cause of osteoporosis was evident in these subjects, whose testosterone levels and gonadal function were normal. Expression analyses showed the presence of RXFP2 in human and mouse osteoblasts. Stimulation of these cells with INSL3 produced a dose- and time-dependent increase in cAMP and cell proliferation, confirming the functionality of the RXFP2/INSL3 receptor-ligand complex. Consistent with the human phenotype, bone histomorphometric and muCT analyses of Rxfp2(-/-) mice showed decreased bone mass, mineralizing surface, bone formation, and osteoclast surface compared with wildtype littermates. This study suggests for the first time a role for INSL3/RXFP2 signaling in bone metabolism and links RXFP2 gene mutations with human osteoporosis.

Strontium ranelate does not stimulate bone formation in ovariectomized rats

Strontium ranelate (SrR) is suggested to function as a dual-acting agent in the treatment of postmenopausal osteoporosis with anti-resorptive and anabolic skeletal benefits. We evaluated the effects of SrR on the skeleton in ovariectomized (OVX) rats and evaluated the influence of dietary calcium. Three-month old virgin female rats underwent ovariectomy (OVX, n = 50) or SHAM surgery (SHAM, n = 10). Four weeks post-surgery, rats were treated daily by oral gavage with distilled water (10 ml/kg/day) or SrR (25 or 150 mg/kg/day) for 90 days. Separate groups of animals for each dose of SrR were fed a low (0.1%) or normal (1.19%) calcium (Ca) diet. Static and dynamic histomorphometry, DXA, mu-CT, mechanical testing, and serum and skeletal concentrations of strontium were assessed. SrR at doses of 25 and 150 mg/kg/day did not increase bone formation on trabecular or periosteal bone surfaces, and failed to inhibit bone resorption of trabecular bone regardless of Ca intake. There were no improvements in bone mass, volume or strength with either dose of SrR given normal Ca. These findings demonstrate that SrR at dosages of 25 and 150 mg/kg/day did not stimulate an anabolic bone response, and failed to improve the bone biomechanical properties of OVX rats.

Bone Disease and Idiopathic Hypercalciuria

Observational and epidemiologic studies alike have shown that idiopathic hypercalciuric (IH) stone-forming patients typically show bone mineral density scores that are significantly lower than those observed for age- and sex-matched normal subjects or those for nonhypercalciuric stone-forming patients. Most of these studies have relied on changes in bone mineral density and have not explored the mechanism(s) involved. There have been a small number of studies that have relied on dynamic bone histomorphometry to ascertain the nature of the bone defect in IH patients. When performed, these studies clearly have shown increased bone resorption and high bone turnover in patients with fasting hypercalciuria whereas suppressed bone formation indices are the most consistent finding in patients with the absorptive variant of IH. The causes of this apparent difference in bone remodeling between the 2 variants of IH still is uncertain. Available evidence suggests that potential mechanisms may be dependent in large part to genetic, metabolic, and nutritional causes of hypercalciuria and bone loss in patients with IH.

Disturbed Synthesis of Type II Collagen Interferes with Rate of Bone Formation and Growth and Increases Bone Resorption in Transgenic Mice

Transgenic mice carrying an internally deleted human type II collagen gene (COL2A1) were used to study bone growth and development. This mutation has previously been shown to disturb the development of collagen fibrils in articular cartilage, causing chondrodysplasia and osteoarthritis. Type II collagen expression in bones was investigated with immunohistochemistry. The development and mineralization of the skeleton and anthropometric measurements on bones were evaluated using X-rays and dynamic histomorphometry. Type II collagen was expressed in the cartilage of developing bones. The bones of transgenic mice were smaller compared with the controls. The bone mass remained almost unchanged in transgenic mice after 1 month of age, leading to differences of 47% in trabecular bone volume (P = 0.012) and 40% in trabecular thickness (P < 0.01) at the age of 3 months compared with controls. At the age of 3 months the eroded surface per bone volume was 31% greater in transgenic mice compared with controls (P < 0.05). Trabecular thickness correlated positively with body weight (R = 0.71, P < 0.001). Interestingly, body weight correlated with bone volume in control mice (R = 0.27, P < 0.01), but no correlation was observed in transgenic mice. The disturbed synthesis of cartilage-specific type II collagen in growing transgenic mice retarded bone development, increased bone resorption, and altered tissue properties. This led to a negative net bone balance and small bone size. The results support the idea that an altered synthesis of cartilage-specific molecule(s) can disturb postnatal bone development and growth.

32 wk old C3H/HeJ mice actively respond to mechanical loading

Numerous studies indicate that C3H/HeJ (C3H) mice are mildly responsive to mechanical loading compared to C57BL/6J (C57) mice. Guided by data indicating high baseline periosteal osteoblast activity in 16 wk C3H mice, we speculated that simply allowing the C3H mice to age until basal periosteal bone formation was equivalent to that of 16 wk C57 mice would restore mechanoresponsiveness in C3H mice. We tested this hypothesis by subjecting the right tibiae of 32 wk old C3H mice and 16 wk old C57 mice to low magnitude rest-inserted loading (peak strain: 1235 μɛ) and then exposing the right tibiae of 32 wk C3H mice to low (1085 μɛ) or moderate (1875 μɛ) magnitude cyclic loading. The osteoblastic response to loading on the endocortical and periosteal surfaces was evaluated via dynamic histomorphometry. At 32 wk of age, C3H mice responded to low magnitude rest-inserted loading with significantly elevated periosteal mineralizing surface, mineral apposition rate and bone formation compared to unloaded contralateral bones. Surprisingly, the periosteal bone formation induced by low magnitude rest-inserted loading in C3H mice exceeded that induced in 16 wk C57 mice. At 32 wk of age, C3H mice also demonstrated an elevated response to increased magnitudes of cyclic loading. We conclude that a high level of basal osteoblast function in 16 wk C3H mice appears to overwhelm the ability of the tissue to respond to an otherwise anabolic mechanical loading stimulus. However, when basal surface osteoblast activity is equivalent to that of 16 wk C57 mice, C3H mice demonstrate a clear ability to respond to either rest-inserted or cyclic loading.

Effects of long-term estrogen replacement therapy on bone turnover in periarticular tibial osteophytes in surgically postmenopausal cynomolgus monkeys

The aims of the present study were to assess the effects of long-term estrogen replacement therapy (ERT) on size and indices of bone turnover in periarticular osteophytes in ovariectomized cynomolgus monkeys and to compare dynamic indices of bone turnover in osteophyte bone with those of subchondral bone (SCB) and epiphyseal/metaphyseal cancellous (EMC) bone. One hundred sixty-five adult female cynomolgus macaques were bilaterally ovariectomized and randomly divided into three age- and weight-matched treatment groups for a 36-month treatment period. Group 1 (OVX control) received no treatment, Group 2 (SPE) received soy phytoestrogens, and Group 3 (ERT) received conjugated equine estrogens in the diet; all monkeys were labeled with calcein before necropsy. A midcoronal, plastic-embedded section of the right proximal tibia from 20 randomly selected animals per treatment group was examined histologically. Forty-nine of the sections (OVX control, n = 16; SPE, n = 16; ERT, n = 17) contained lateral abaxial osteophytes, and static and dynamic histomorphometry measurements were taken from osteophyte bone, SCB from the lateral tibial plateau, and EMC bone. Data were analyzed using the ANOVA and Kruskal–Wallis test, correlation and regression methods, and the Friedman and Wilcoxon signed rank test. There was no significant effect of long-term ERT on osteophyte area or on any static or dynamic histomorphometry parameters. The bone volume, trabecular number, and trabecular thickness in osteophyte bone were considerably higher than in EMC bone; whereas, trabecular separation was considerably lower in osteophyte bone. In all three treatment groups, BS/BV was significantly lower in osteophyte bone vs. EMC bone and significantly higher in osteophyte bone vs. lateral SCB. We conclude that osteophyte area and static and dynamic histomorphometry parameters within periarticular tibial osteophytes in ovariectomized cynomolgus monkeys are not significantly influenced by long-term ERT, but that site differences in static and dynamic bone histomorphometry parameters exist, particularly between EMC and osteophyte bone.

Wnt10b Increases Postnatal Bone Formation by Enhancing Osteoblast Differentiation

Overexpression of Wnt10b from the osteocalcin promoter in transgenic mice increases postnatal bone mass. Increases in osteoblast perimeter, mineralizing surface, and bone formation rate without detectable changes in pre-osteoblast proliferation, osteoblast apoptosis, or osteoclast number and activity suggest that, in this animal model, Wnt10b primarily increases bone mass by stimulating osteoblastogenesis. Wnt signaling regulates many aspects of development including postnatal accrual of bone. Potential mechanisms for how Wnt signaling increases bone mass include regulation of osteoblast and/or osteoclast number and activity. To help differentiate between these possibilities, we studied mice in which Wnt10b is expressed specifically in osteoblast lineage cells or in mice devoid of Wnt10b. Transgenic mice, in which mouse Wnt10b is expressed from the human osteocalcin promoter (Oc-Wnt10b), were generated in C57BL/6 mice. Transgene expression was evaluated by RNase protection assay. Quantitative assessment of bone variables was done by radiography, muCT, and static and dynamic histomorphometry. Mechanisms of bone homeostasis were evaluated with assays for BrdU, TUNEL, and TRACP5b activity, as well as serum levels of C-terminal telopeptide of type I collagen (CTX). The endogenous role of Wnt10b in bone was assessed by dynamic histomorphometry in Wnt10b(-/-) mice. Oc-Wnt10b mice have increased mandibular bone and impaired eruption of incisors during postnatal development. Analyses of femoral distal metaphyses show significantly higher BMD, bone volume fraction, and trabecular number. Increased bone formation is caused by increases in number of osteoblasts per bone surface, rate of mineral apposition, and percent mineralizing surface. Although number of osteoclasts per bone surface is not altered, Oc-Wnt10b mice have increased total osteoclast activity because of higher bone mass. In Wnt10b(-/-) mice, changes in mineralizing variables and osteoblast perimeter in femoral distal metaphyses were not observed; however, bone formation rate is reduced because of decreased total bone volume and trabecular number. High bone mass in Oc-Wnt10b mice is primarily caused by increased osteoblastogenesis, with a minor contribution from elevated mineralizing activity of osteoblasts.

Renal Osteodystrophy After Successful Renal Transplantation: A Histomorphometric Analysis in 57 Patients

Renal transplantation is the treatment of choice for patients with end-stage renal disease. It corrects most of the metabolic abnormalities that cause renal osteodystrophy. Nevertheless, renal osteodystrophy persists in many transplant recipients. The aim of this study was to investigate frequency and histomorphometric pattern of bone disease after renal transplantation. Bone biopsy specimens were taken from the iliac crest of 57 patients, including 28 women (26–70 years old) and 29 men (27–67 years old). Indications for biopsy were hypercalcemia, elevation of parathyroid hormone, and, in 19 cases, without suspected bone abnormalities based on laboratory parameters. The mean time of dialysis prior to renal transplantation was 43 months (range, 6–91 months in women and 10–111 months in men) and the mean interval between transplantation and bone biopsy was 53.5 months (range, 4–191 months in women and 5–90 months in men). Fourteen patients were treated with either 25-hydroxyvitamin D 3 and/or 1-α hydroxyvitamin D 3 or 1,25 dihydroxyvitamin D 3, 3 with phosphate-binding agents. The immunosuppression consisted of cyclosporine, azathioprine, and prednisolone. The cumulative dosage of corticosteroids was 5569 ± 5305 mg. For static and dynamic histomorphometry, we used American Society of Bone and Mineral Research nomenclature. Mild osteitis fibrosa and osteitis fibrosa, the most frequent forms of renal osteodystrophy, were observed in 13. (22.8%) and 14 patients (24.6%), respectively. Mixed uremic osteodystrophy was found in 7 patients (12.3%), adynamic renal bone disease in 3 patients (5.3%), and osteomalacia in 2 patients (3.5%). In 13 patients (22.8%), reduced bone mass and structural damage without typical signs of renal osteodystrophy, such as endosteal fibrosis or osteoclasia, were detected, and 5 patients (8.7%) showed normal histomorphometric parameters. We concluded that renal osteodystrophy, especially forms with high bone turnover, persisted in many patients after successful renal transplantation. This finding may be due to preexisting conditions, such as duration of dialysis and degree of hyperparathyroidism. Bone disease is increased by corticosteroid and immunosuppressive therapy after renal transplantation and requires close monitoring.

Prospective Evaluation of the Bone Anabolic Effect of Bortezomib in Relapsed Multiple Myeloma (MM) Patients.

Bortezomib (B) has been shown to increase osteoblast activity and inhibit osteoclast formation, thus promoting bone formation. We have previously reported a correlation between increased alkaline phosphatase (ALP) and the response to B in patients with MM. We now report results of a detailed prospective study examining the skeletal effects of B treatment in MM patients using histomorphometry, micro computed tomography (microCT) and markers of bone metabolism.Methods: Single agent B (1.3 mg/m2 patients 1–10; 1mg/m2 patient 11–20), was administered on days 1, 4, 8 and 11 on a 21 day intervals for a total of 3 cycles; no patients were receiving concurrent bisphosphonates or steroids during the entire study period. The dynamic indices of bone turnover were prospectively evaluated via tetracycline labeling prior to transiliac bone biopsies obtained at baseline and after 3 cycles of treatment. Biopsy specimens were examined by high-resolution microCT prior to histomorphometric analyses. Architectural parameters such as bone volume/total volume (BVTV), trabecular number (TbN), and thickness (Tb.Th) were recorded. Osteocalcin, and alkaline phosphatase on days 1,4,8,11 were measured before and after each B dose and every 4 hours thereafter, daily for the other days of the treatment cycle.Results: Histomorphometric analyses were compared in 7 of the 11 patients who completed the trial. All 11 patients underwent bone biopsy at baseline nine of those 11 at the end of the study (2 samples were not adequate). Baseline BV/TV values ranged from 13% to 80%. After 3 cycles of B treatment % increase (mean 37%) in BV/TV was shown in 6 of 7 patients (P<0.034). Tb.Th was also increased from baseline (range 20%–456%) in 5 of 7 patients (71%) who responded to B. Histological bone histomorphometry demonstrated a lack of osteoid formation and osteoblast activity at baseline and a marked increased in osteoid and osteoblast numbers on trabecular and cortical bone surface following B treatment. Tetracycline incorporation into bone was observed in only 2 of 11 of patients (18%) at baseline, reflecting the extremely low levels of bone turnover in MM patients prior to treatment. However, post-B tetracycline labeling was observed in 7 of 11 (63%) samples (p<0.03; baseline vs post treatment score changes), reflecting the dramatic increases in bone turnover elicited by B treatment. Interestingly, after B treatment, the rate of bone formation was accelerated in the 2 patients with measurable baseline tetracycline labeling. Baseline osteocalcin values were below the reference intervals (11–50 ng/ml) in 10 of 11 patients but increased in 9 of 11 patients at the end of the third B cycle. Overall osteocalcin changes increased from baseline by 403% (p<0.037). BALP ranged from 4.5 to 48.4 ug/l at baseline and increased in 6 of 10 patients following B treatment.Conclusion: In this first prospective single agent B study we demonstrate that even a short course (3 cycles or 12 doses) of B treatment has a significant and potent anabolic bone effect in MM patients, demonstrated by serum turnover markers, micro-CT measures of bone architecture and by static and dynamic histomorphometry.

In Vivo Effects of Zoledronic Acid on Bone Remodeling.

Zoledronic acid (ZA) is an amino-bisphosphonate with very potent antiresorptive activity widely used in the treatment of multiple myeloma (MM) bone disease. Recently, the increasing incidence of osteonecrosis of the jaw and its possible association with prolonged use of amino-bisphosphonates such as ZA has been reported. Therefore, we here studied the effects of ZA on bone remodeling in vitro and in vivo. Although ZA has been shown to inhibit osteoclastogenesis and increase bone mineralization, data on its effects on osteoblastic activity and on bone formation are conflicting. To study the effects of ZA on osteoblasts (OB), 5 week old C57BL/6 mice were treated with low and high doses of ZA intraperitoneally (IP) weekly. The dose ranged from 0.05-1mg/kg, with the highest dose recapitulating a lifetime dose of ZA over a 5 year period in an adult MM patient. Blood was collected at baseline and weekly thereafter. IP calcein injections were administered to study bone formation rates. Consistent with its known effects on bone mass and density, in vivo DXA scans in ZA-treated animals demonstrated an increase in whole body bone mineral density (BMD). ZA treatment was associated with a dose-related increase in trabecular bone at the distal femur, evaluated by microCT and confirmed by static histomorphometry. ELISA assays showed a decrease in TRACP5B (bone resorption), as expected due to the anti-osteoclastic activity of ZA. In addition to osteoclast (OC) inhibition, mice treated with ZA also showed alterations in OB activity. Specifically, serum osteocalcin (a marker for bone formation) levels were lower in ZA treated mice, and dynamic histomorphometry confirmed decreased bone formation rates. In order to study the mechanism of our in vivo observations, we tested the effects of ZA on OB, OC, and bone marrow stromal cells (BMSCs) treated in vitro with ZA (0.01μM to 100μM) for 7, 14, and 21 days. Decreased viability during differentiation of both OB and OC was observed, without any significant effects on BMSCs. This was associated with decreased alkaline phosphatase activity, suggesting functional impairment of OB activity. Our data therefore suggests that ZA impairs OB number and activity, in addition to effects on OC and may impair normal physiologic bone remodeling. Ongoing studies will determine the molecular mechanisms whereby ZA mediates these sequelae and inform future studies of ZA use in patients with MM bone disease.

Spinal cord injury causes rapid osteoclastic resorption and growth plate abnormalities in growing rats (SCI-induced bone loss in growing rats)

Spinal cord injury causes severe bone loss. We report osteoclast resorption with severe trabecular and cortical bone loss, decreased bone mineral apposition, and growth plate abnormalities in a rodent model of contusion spinal cord injury. These findings will help elucidate the mechanisms of osteoporosis following neurological trauma. Limited understanding of the mechanism(s) that underlie spinal cord injury (SCI)-induced bone loss has led to few treatment options. As SCI-induced osteoporosis carries significant morbidity and can worsen already profound disability, there is an urgency to advance knowledge regarding this pathophysiology. A clinically relevant contusion model of experimental spinal cord injury was used to generate severe lower thoracic SCI by weight-drop (10 g x 50 mm) in adolescent male Sprague-Dawley rats. Body weight and gender-matched naïve (no surgery) rats served as controls. Bone microarchitecture was determined by micro-computed tomographic imaging. Mature osteoclasts were identified by TRAP staining and bone apposition rate was determined by dynamic histomorphometry. At 10 days post-injury we detected a marked 48% decrease in trabecular bone and a 35% decrease in cortical bone at the distal femoral metaphysis by micro-CT. A 330% increase in the number of mature osteoclasts was detected at the growth plate in the injured animals that corresponded with cellular disorganization at the chondro-osseous junction. Appositional growth studies demonstrated decreased new bone formation with a mineralization defect indicative of osteoblast dysfunction. Contusion SCI results in a rapid bone loss that is the result of increased bone resorption and decreased bone formation.

Three Years of Alendronate Treatment Results in Similar Levels of Vertebral Microdamage as After One Year of Treatment

Three years of daily alendronate treatment increases microdamage in vertebral bone but does not significantly increase it beyond levels of microdamage found after 1 yr of treatment. This suggests microdamage accumulation peaks during the early period of bisphosphonate treatment and does not continue to accumulate with longer periods of treatment. Clinically relevant doses of alendronate increase vertebral microdamage by 4- to 5-fold in skeletally mature beagles after 1 yr of treatment. The goal of this study was to determine whether microdamage would continue to accumulate with 3 yr of alendronate treatment in an intact beagle dog model. One-year-old female beagles were treated with daily oral doses of vehicle (VEH, 1 ml/kg/d) or alendronate (ALN, 0.2 or 1.0 mg/kg/d) for 3 yr. These ALN doses were chosen to approximate, on a milligram per kilogram basis, those used to treat osteoporosis (ALN0.2) and Paget's disease (ALN1.0). Microdamage accumulation, static and dynamic histomorphometry, densitometry, and mechanical properties of lumbar vertebrae were assessed. Comparisons were made among the three groups treated for 3 yr and also within each treatment group to animals treated under the same conditions for 1 yr. Overall microdamage accumulation (crack surface density) was not significantly higher in animals treated for 3 yr with either dose of ALN, whereas crack density increased significantly (100%; p < 0.05) with the higher dose of ALN compared with VEH. Both ALN doses significantly suppressed the rate of bone turnover (-60% versus VEH). There was no difference among groups for any of the structural biomechanical properties-ultimate load, stiffness, or energy absorption. However, when adjusted for areal BMD, ALN-treated animals had significantly lower energy absorption (-20%) compared with VEH. Toughness, the energy absorption capacity of the bone tissue, was significantly lower than VEH for both ALN0.2 (-27%) and ALN1.0 (-33%). Compared with animals treated for 1 yr, there was no significant difference in microdamage accumulation for either ALN dose. VEH-treated animals had significantly lower bone turnover (-58%) and significantly higher levels of microdamage (+300%) compared with values in 1-yr animals. Toughness was significantly lower in animals treated for 3 yr with ALN1.0 (-18%) compared with animals treated for 1 yr, whereas there was no difference in toughness between the two treatment durations for either VEH or ALN0.2. Although 3 yr of ALN treatment resulted in higher microcrack density in vertebral trabecular bone compared with control dogs, the amount of microdamage was not significantly higher than animals treated for 1 yr with similar doses. This suggests that bisphosphonate-associated increases in microdamage occur early in treatment. Because toughness continued to decline significantly over 3 yr of treatment at the higher ALN dose, decreases in toughness are probably not dependent on damage accumulation.