ß-Arrestin 2 knockout prevents bone loss in response to continuous parathyroid hormone stimulation in male and female mice.

BACKGROUND: ß-Arrestin 2 (ß-arr2) binds activated parathyroid hormone (PTH) receptors stimulating internalization. PTH stimulates both anabolic and catabolic effect on bone depending on the way it is administered. Intermittent PTH stimulation increases trabecular bone formation in mice, but this is decreased in mice lacking ß-arr 2, suggesting a role for ß-arr 2 in the anabolic effects of PTH. The role of ß-arr 2 in the catabolic effects of continuous PTH (cPTH) treatment is not known. OBJECTIVE: To assess the effects of cPTH administration on bone in mice lacking ß-arr 2 compared to wild-type (WT). METHODS: Groups of male and female WT or ß-arr2 knockout (KO) mice were administered either PTH or phosphate-buffered saline by osmotic pumps for 2 weeks. Following treatment, serum calcium and phosphate levels were measured, bone structure and mineral density were measured by microcomputed tomography, and bone cells measured by static and dynamic histomorphometry. RESULTS: ß-arr2 KO had no effects on skeletal development in mice of either sex. PTH treatment caused hypercalcemia and hypophosphatemia and decreased trabecular and cortical bone only in male WT mice. ß-arr2 KO in male mice completely abrogated the effects of PTH on bone, while in female ß-arr2 KO mice, PTH treatment increased trabecular bone with no effects on cortical bone. CONCLUSIONS: These results demonstrate a profound sex effect on skeletal responses to cPTH treatment, suggesting a protective effect of estrogen on bone loss. ß-arr2 plays a role in restraining the anabolic effects of PTH in both male and female mice.

Chlorthalidone with potassium citrate decreases calcium oxalate stones and increases bone quality in genetic hypercalciuric stone-forming rats.

To study human idiopathic hypercalciuria we developed an animal model, genetic hypercalciuric stone-forming rats, whose pathophysiology parallels that of human idiopathic hypercalciuria. Fed the oxalate precursor, hydroxyproline, every rat in this model develops calcium oxalate stones. Using this rat model, we tested whether chlorthalidone and potassium citrate combined would reduce calcium oxalate stone formation and improve bone quality more than either agent alone. These rats (113 generation) were fed a normal calcium and phosphorus diet with hydroxyproline and divided into four groups: diets plus potassium chloride as control, potassium citrate, chlorthalidone plus potassium chloride, or potassium citrate plus chlorthalidone. Urine was collected at six, 12, and 18 weeks and kidney stone formation and bone parameters were determined. Compared to potassium chloride, potassium citrate reduced urinary calcium, chlorthalidone reduced it further and potassium citrate plus chlorthalidone even further. Potassium citrate plus chlorthalidone decreased urine oxalate compared to all other groups. There were no significant differences in calcium oxalate supersaturation in any group. Neither potassium citrate nor chlorthalidone altered stone formation. However, potassium citrate plus chlorthalidone significantly reduced stone formation. Vertebral trabecular bone increased with chlorthalidone and potassium citrate plus chlorthalidone. Cortical bone area increased with chlorthalidone but not potassium citrate or potassium citrate plus chlorthalidone. Mechanical properties of trabecular bone improved with chlorthalidone, but not with potassium citrate plus chlorthalidone. Thus in genetic hypercalciuric stone-forming rats fed a diet resulting in calcium oxalate stone formation, potassium citrate plus chlorthalidone prevented stone formation better than either agent alone. Chlorthalidone alone improved bone quality, but adding potassium citrate provided no additional benefit.

Ontogenetic changes to bone microstructure in an archaeologically derived sample of human ribs.

There is considerable variation in the gross morphology and tissue properties among the bones of human infants, children, adolescents, and adults. Using 18 known-age individuals (nfemale  = 8, nmale  = 9, nunknown  = 1; birth to 21 years old), from a well-documented cemetery collection, Spitalfields Christ Church, London, UK, this study explores growth-related changes in cortical and trabecular bone microstructure. Micro-CT scans of mid-shaft middle thoracic ribs are used for quantitative analysis. Results are then compared to previously quantified conventional histomorphometry of the same sample. Total area (Tt.Ar), cortical area (Ct.Ar), cortical thickness (Ct.Th), and the major (Maj.Dm) and minor (Min.Dm) diameters of the rib demonstrate positive correlations with age. Pore density (Po.Dn) increases, but age-related changes to cortical porosity (Ct.Po) appear to be non-linear. Trabecular thickness (Tb.th) and trabecular separation (Tb.Sp) increase with age, whereas trabecular bone pattern factor (Tb.Pf), structural model index (SMI), and connectivity density (Conn.D) decrease with age. Sex-based differences were not identified for any of the variables included in this study. Some samples display clear evidence of diagenetic alteration without corresponding changes in radiopacity, which compromises the reliability of bone mineral density (BMD) data in the study of past populations. Cortical porosity data are not correlated with two-dimensional measures of osteon population density (OPD). This suggests that unfilled resorption spaces contribute more significantly to cortical porosity than do the Haversian canals of secondary osteons. Continued research using complementary imaging techniques and a wide array of histological variables will increase our understanding of age- and sex-specific ontogenetic patterns within and among human populations.

Chlorthalidone Is Superior to Potassium Citrate in Reducing Calcium Phosphate Stones and Increasing Bone Quality in Hypercalciuric Stone-Forming Rats.

BACKGROUND: The pathophysiology of genetic hypercalciuric stone-forming rats parallels that of human idiopathic hypercalciuria. In this model, all animals form calcium phosphate stones. We previously found that chlorthalidone, but not potassium citrate, decreased stone formation in these rats. METHODS: To test whether chlorthalidone and potassium citrate combined would reduce calcium phosphate stone formation more than either medication alone, four groups of rats were fed a fixed amount of a normal calcium and phosphorus diet, supplemented with potassium chloride (as control), potassium citrate, chlorthalidone (with potassium chloride to equalize potassium intake), or potassium citrate plus chlorthalidone. We measured urine every 6 weeks and assessed stone formation and bone quality at 18 weeks. RESULTS: Potassium citrate reduced urine calcium compared with controls, chlorthalidone reduced it further, and potassium citrate plus chlorthalidone reduced it even more. Chlorthalidone increased urine citrate and potassium citrate increased it even more; the combination did not increase it further. Potassium citrate, alone or with chlorthalidone, increased urine calcium phosphate supersaturation, but chlorthalidone did not. All control rats formed stones. Potassium citrate did not alter stone formation. No stones formed with chlorthalidone, and rats given potassium citrate plus chlorthalidone had some stones but fewer than controls. Rats given chlorthalidone with or without potassium citrate had higher bone mineral density and better mechanical properties than controls, whereas those given potassium citrate did not. CONCLUSIONS: In genetic hypercalciuric stone-forming rats, chlorthalidone is superior to potassium citrate alone or combined with chlorthalidone in reducing calcium phosphate stone formation and improving bone quality.

Pre-treatment with Pamidronate Improves Bone Mechanical Properties in Mdx Mice Treated with Glucocorticoids.

Duchenne muscular dystrophy (DMD) is an X-linked disease of progressive muscle deterioration and weakness. Patients with DMD have poor bone health which is partly due to treatment with glucocorticoids, a standard therapy to prolong muscle function that also induces bone loss. Bisphosphonates are used to treat adults at risk of glucocorticoid-induced osteoporosis but are not currently used in DMD patients until after they sustain fractures. In this study, C57BL/10ScSn-mdx mice, a commonly used DMD animal model, received continuous glucocorticoid, prednisone treatment (0.083 mg/day) from 5 to 10 weeks of age. Pre-treatment with the bisphosphonate pamidronate started at 4 weeks of age over a period of 2 weeks or 6 weeks (cumulative dose 8 mg/kg for both) to assess the effectiveness of the two dosing regimens in ameliorating glucocorticoid-induced bone loss. Mdx mice treated with prednisone had improved muscle function that was not changed by pamidronate treatment. Glucocorticoid treatment caused cortical bone loss and decreased cortical bone strength. Both 2 and 6 week pamidronate treatment increased cortical thickness and bone area compared to prednisone-treated Mdx mice, however, only 2 week pamidronate treatment improved the strength of cortical bone compared to that of glucocorticoid-treated Mdx mice. In the trabecular bone, both pamidronate treatments significantly increased the amount of bone, and increased the ultimate load but not the energy to fail. These results highlight the importance of when and how much bisphosphonate is administered prior to glucocorticoid exposure.

Use of backscattered scanning electron microscopy to quantify the bone tissues of midthoracic human ribs.

OBJECTIVES: Novel information on apartheid health conditions may be obtained through the study of recent skeletal collections. Using a backscattered scanning electron microscopy (BSE-SEM) approach, this study aims to produce bone quality and tissue mineralization data for an understudied South African population from the Western Cape province. METHODS: Using BSE-SEM imaging, cortical porosity (Ct.Po), osteocyte lacunar density (Ot.Lc.Dn), and the degree of tissue mineralization were quantified in midthoracic ribs from the Kirsten Skeletal Collection. Individuals ( n female = 75, n male = 68, and mean age = 46.3 years) were predominantly from the South Africa Colored (SAC) population group ( n SAC = 103, 72%). Full cross-sectional images of each rib were manually stitched together in Adobe Photoshop. Photomontages were imported into MATALB (Mathworks, Natick, MA) for image processing and analysis. Age-related changes in histomorphometric parameters and sex differences were examined using correlation analysis, as well as linear and nonlinear regressions. RESULTS: Young adult men have significantly less mineralized bone and fewer osteocyte lacunae, compared to women. Only men demonstrate a significant negative relationship between Ot.Lc.Dn and age. Average tissue mineralization decreases with age in women, while Ct.Po increases. Pore area (Po.Ar) does not vary with age, but pore density (Po.Dn) is highest in the perimenopause, when accelerated rates of bone turnover are first anticipated. Ct.Po is highest in the years following the predicted age of menopause, but levels off in the final decades of life. CONCLUSIONS: Men and women display disparate patterns of bone aging. Systemic disenfranchisement of non-white population groups affected bone health in South Africa, and may continue to do so today. Indicators of poor bone quality are evident in the full study sample, indicating that osteoporosis and fracture risk are not just of concern to the aged white female population.

Ribose pre-treatment can protect the fatigue life of γ-irradiation sterilized bone.

Structural bone allografts are often sterilized with γ-irradiation to decrease infection risk, which unfortunately degrades the bone collagen connectivity, making the bone weak and brittle. In previous studies, we successfully protected the quasi-static mechanical properties of human cortical bone by pre-treating with ribose, prior to irradiation. This study focused on the quasi-static and fatigue tensile properties of ribose treated irradiated sterilized bone allografts. Seventy-five samples were cut from the mid-shaft diaphysis of human femurs into standardized dog-bone shape geometries for quasi-static and fatigue tensile testing. Specimens were prepared in sets of three adjacent specimens. Each set was made of a normal (N), irradiated (I) and ribose pre-treated + irradiation (R) group. The R group was incubated in a 1.2 M ribose solution before γ-irradiation. The quasi-static tensile and decalcified tests were conducted to failure under displacement control. The fatigue samples were tested under cyclic loading (10 Hz, peak stress of 45MP, minimum-to-maximum stress ratio of 0.1) until failure or reaching 10 million cycles. Ribose pre-treatment significantly improved significantly the mechanical properties of irradiation sterilized human bone in the quasi-static tensile and decalcified tests. The fatigue life of the irradiated group was impaired by 99% in comparison to the normal control. Surprisingly, the R-group has significantly superior properties over the I-group and N-group (p < 0.01, p < 0.05) (> 100%). This study shows that incubating human cortical bone in a ribose solution prior to irradiation can indeed improve the fatigue life of irradiation-sterilized cortical bone allografts.

Effect of 25-HydroxyVitamin D Deficiency and Its Interaction with Prednisone Treatment on Musculoskeletal Health in Growing Mdx Mice.

Duchenne muscular dystrophy (DMD) results from genetic mutations of the gene encoding dystrophin, leading to muscle inflammation and degeneration that is typically treated with glucocorticoids. DMD and its treatment with glucocorticoids result in poor bone health and high risk of fractures. Insufficient levels of 25-hydroxyvitamin D (25-hydroxy D) that may contribute to weakened bone are routinely found in DMD patients. To determine the effect of 25-hydroxy D deficiency, this study examined the effects of low vitamin D dietary intake with and without glucocorticoids on the musculoskeletal system of the Mdx mouse model of DMD. At 10 weeks of age, Mdx mice on control diet had low trabecular bone mineral density of distal femurs and lumbar vertebrae with increased osteoclast numbers compared to wild-type mice. Low vitamin D intake resulted in 25-hydroxy D deficiency but had no effect on trabecular or cortical bone. Cortical bone loss and bone weakness were induced by glucocorticoids while they improved muscle grip strength in Mdx mice. 25-hydroxy D deficiency did not result in any significant effects on growing bone or muscle in the Mdx mice. In combination with glucocorticoid treatment, low 25-hydroxy D resulted in no change in cortical bone mineral density but bone ductility was significantly increased suggesting lower bone mineralization.

Sex-specific patterns in cortical and trabecular bone microstructure in the Kirsten Skeletal Collection, South Africa.

OBJECTIVES: The purpose of this study was to provide bone histomorphometric reference data for South Africans of the Western Cape who likely dealt with health issues under the apartheid regime. METHODS: The 206 adult individuals (n female = 75, n male = 131, mean = 47.9 ± 15.8 years) from the Kirsten Skeletal Collection, U. Stellenbosch, lived in the Cape Town metropole from the late 1960s to the mid-1990s. To study age-related changes in cortical and trabecular bone microstructure, photomontages of mid-thoracic rib cross-sections were quantitatively examined. Variables include relative cortical area (Rt.Ct.Ar), osteon population density (OPD), osteon area (On.Ar), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp). RESULTS: All cortical variables demonstrated significant relationships with age in both sexes, with women showing stronger overall age associations. Peak bone mass was compromised in some men, possibly reflecting poor nutritional quality and/or substance abuse issues throughout adolescence and early adulthood. In women, greater predicted decrements in On.Ar and Rt.Ct.Ar suggest a structural disadvantage with age, consistent with postmenopausal bone loss. Age-related patterns in trabecular bone microarchitecture are variable and difficult to explain. Except for Tb.Th, there are no statistically significant relationships with age in women. Men demonstrate significant negative correlations between BV/TV, Tb.N, and age, and a significant positive correlation between Tb.Sp and age. CONCLUSIONS: This research highlights sex-specific differences in patterns of age-related bone loss, and provides context for discussion of contemporary South African bone health. While the study sample demonstrates indicators of poor bone quality, osteoporosis research continues to be under-prioritized in South Africa.

The Lyme Disease Pathogen Borrelia burgdorferi Infects Murine Bone and Induces Trabecular Bone Loss.

Lyme disease is caused by members of the Borrelia burgdorferi sensu lato species complex. Arthritis is a well-known late-stage pathology of Lyme disease, but the effects of B. burgdorferi infection on bone at sites other than articular surfaces are largely unknown. In this study, we investigated whether B. burgdorferi infection affects bone health in mice. In mice inoculated with B. burgdorferi or vehicle (mock infection), we measured the presence of B. burgdorferi DNA in bones, bone mineral density (BMD), bone formation rates, biomechanical properties, cellular composition, and two- and three-dimensional features of bone microarchitecture. B. burgdorferi DNA was detected in bone. In the long bones, increasing B. burgdorferi DNA copy number correlated with reductions in areal and trabecular volumetric BMDs. Trabecular regions of femora exhibited significant, copy number-correlated microarchitectural disruption, but BMD, microarchitectural, and biomechanical properties of cortical bone were not affected. Bone loss in tibiae was not due to increased osteoclast numbers or bone-resorbing surface area, but it was associated with reduced osteoblast numbers, implying that bone loss in long bones was due to impaired bone building. Osteoid-producing and mineralization activities of existing osteoblasts were unaffected by infection. Therefore, deterioration of trabecular bone was not dependent on inhibition of osteoblast function but was more likely caused by blockade of osteoblastogenesis, reduced osteoblast survival, and/or induction of osteoblast death. Together, these data represent the first evidence that B. burgdorferi infection induces bone loss in mice and suggest that this phenotype results from inhibition of bone building rather than increased bone resorption.

The effect of ribose pre-treatment of cortical bone on γ-irradiation sterilization effectiveness.

Reconstruction of large skeletal defects is a significant and challenging issue. Tissue banks often use γ-irradiation (15-35 kGy) to sterilize bone allografts, which, however, drastically impairs the post-yield mechanical properties. In previous studies, we reported the development of a method that protects human bone collagen connectivity through ribose crosslinking while still undergoing γ-irradiation. Given these promising results, the next step was to determine if the presence of ribose within the bone tissue would interfere with the effectiveness of the γ-irradiation sterilization against bacteria. This study had two stages. The aim of the first stage was to assess the protective effect of ribose in solution using a Bacillus pumilus spore strip model. The aim of the second stage was to assess the protective effect of ribose (R) on spores within a human cortical bone model in comparison to conventionally irradiated bone (I). Treatment of B. pumilus spore strips with ribose in solution led to temperature-dependent effects on spore viability versus spore strips treated with PBS alone. Ribose solution at 60 °C led to a notable two logs decrease in spore count relative to PBS at 60 °C. In the human bone model, the number of spores in the I and R groups were greatly decreased in comparison to the non-irradiated N group. No spore colonies were detected in the R group (n = 4) whereas two of the four plates of group I formed colonies. This study provides evidence that the method of pre-treating bone with ribose crosslinking prior to irradiation sterilization, while improving irradiation sterilized bone allograft quality, also may improve the effectiveness of the sterilization process.

Development of a novel method for the strengthening and toughening of irradiation-sterilized bone allografts.

Reconstruction of large skeletal defects is a significant and challenging issue. Bone allografts are often used for such reconstructions. However, sterilizing bone allografts by using γ-irradiation, damages collagen and causes the bone to become weak, brittle and less fatigue resistant. In a previous study, we successfully protected the mechanical properties of human cortical bone by conducting a pre-treatment with ribose, a natural and biocompatible agent. This study focuses on examining possible mechanisms by which ribose might protect the bone. We examined the mechanical properties, crosslinking, connectivity and free radical scavenging potentials of the ribose treatment. Human cortical bone beams were treated with varying concentration of ribose (0.06-1.2 M) and γ-irradiation before testing them in 3-point bending. The connectivity and amounts of crosslinking were determined with Hydrothermal-Isometric-Tension testing and High-Performance-Liquid-Chromatography, respectively. The free radical content was measured using Electron Paramagnetic Resonance. Ribose pre-treatment improved the mechanical properties of irradiation sterilized human bone in a pre-treatment concentration-dependent manner. The 1.2 M pre-treatment provided >100% of ultimate strength of normal controls and protected 76% of the work-to-fracture (toughness) lost in the irradiated controls. Similarly, the ribose pre-treatment improved the thermo-mechanical properties of irradiation-sterilized human bone collagen in a concentration-dependent manner. Greater free radical content and pentosidine content were modified in the ribose treated bone. This study shows that the mechanical properties of irradiation-sterilized cortical bone allografts can be protected by incubating the bone in a ribose solution prior to irradiation.

Elevated Gα11 expression in osteoblast lineage cells promotes osteoclastogenesis and leads to enhanced trabecular bone accrual in response to pamidronate.

Osteoblastic cells indirectly induce osteoclastogenesis in the bone microenvironment by expressing paracrine factors such as RANKL and M-CSF, leading to increased bone resorption. These cytokines can be regulated by a variety of intracellular pathways, which include G protein-coupled receptor signaling. To explore how enhanced signaling of the Gαq/11 pathway in osteoblast lineage cells may mediate osteoclast formation, we cocultured wild-type (WT) preosteoclasts with BMSCs derived from either WT or transgenic mice with osteoblast-specific overexpression of Gα11 (G11-Tg). G11-Tg cocultures had elevated osteoclast numbers with greater resorptive capacity and increased expression of Rankl, Rankl:Opg (osteoprotegerin), and M-csf compared with cocultures with WT BMSCs. As well, cocultures with G11-Tg BMSCs required a higher concentration of OPG to inhibit osteoclast formation and less angiotensin II to increase osteoclast size. These indicate that G11-Tg osteoblasts drive the increased osteoclast formation and osteopenia seen in G11-Tg mice. Pamidronate treatment of G11-Tg mice restored the trabecular bone loss phenotype, as bone mineral density, bone volume, trabecular number, separation, and expressions of osteoblastic and osteoclastic genes were comparable with WT parameters. These changes were characterized by enhanced accumulation of calcified cartilage in trabecular bone, demonstrating that resorption of the cartilaginous intermediate by osteoclasts is more affected by bisphosphonate treatment in G11-Tg mice. In conclusion, overexpression of Gα11 in osteoblastic cells promotes osteoclastogenesis by upregulation of Rankl and M-csf and bone loss by increased osteoclast resorption of the trabecular bone and cartilaginous matrix.

Overexpression of Gα11 in Osteoblast Lineage Cells Suppresses the Osteoanabolic Response to Intermittent PTH and Exercise.

Intermittent parathyroid hormone (iPTH) treatment and mechanical loading are osteoanabolic stimuli that are partially mediated through actions on G protein-coupled receptors (GPCRs). GPCR signaling can be altered by heterotrimeric G protein Gα subunits levels, which can therefore lead to altered responses to such stimuli. Previous studies have suggested that enhanced signaling through the Gαq/11 pathway inhibits the osteoanabolic actions of PTH. The influence of Gαq/11 signaling on mechanotransduction, however, has not been reported in vivo. Using transgenic mice that specifically overexpress Gα11 in osteoblast lineage cells (G11-Tg mice), we investigated the skeletal effects of elevated Gα11 levels on iPTH and mechanical loading by treadmill exercise. Both regimens increased trabecular and cortical bone in Wild-Type (WT) mice as a result of increased bone formation. In G11-Tg mice, there was no change in trabecular or cortical bone and no increase in bone formation in response to iPTH or exercise. While exercise reduced osteoclast parameters in WT mice, these changes were diminished in G11-Tg mice as expression of M-csf and Trap remained increased. Collectively, our results suggest that osteoblastic upregulation of Gα11 is inhibitory to osteoanabolic actions of both PTH and exercise, and that its suppression may be a promising target for treating bone loss.

Long-term effects of castration on the skeleton of male rhesus monkeys (Macaca mulatta).

While osteopenia (OPE) and osteoporosis (OPO) have been studied in various species of aging nonhuman primates and extensively in ovariectomized rhesus and cynomolgus macaques, there is virtually no information on the effects of castration on the skeleton of male nonhuman primates. Most information on castrated male primates comes from a few studies on the skeletons of eunuchs. This report used a subset of the Caribbean Primate Research Center's (CPRC) Cayo Santiago (CS) rhesus macaque skeletal collection to qualitatively and quantitatively compare the bone mineral density (BMD) of castrated and age-matched intact males and, thereby, determine the long-term effects of castration (orchidectomy) on bone. Lumbar vertebrae, femora, and crania were evaluated using dual-energy X-ray absorptiometry (DEXA or DXA) and digital radiography augmented, when fresh tissues were available, with autoradiography and histology. Results confirmed physical examinations of long bones that castration causes changes in the skeleton of male rhesus macaques similar to those found in eunuchs, including OPE and OPO of the vertebrae and femora, thinning of the skull, and vertebral fractures and kyphosis of the spine more severe than that caused by normal aging alone. Also like eunuchs, some castrated CS male rhesus monkeys had a longer life span than intact males or females. Based on these results and the effects of castration on other tissues and organs of eunuchs, on behavior, hormone profiles and possibly on cognition and visual perception of human and nonhuman primates, and other mammals, castrated male rhesus macaques should be used with caution for laboratory studies and should be considered a separate category from intact males. Despite these caveats, the castrated male rhesus macaque should make an excellent animal model in which to test hormone replacement therapies for boys and men orchidectomized for testicular and prostate cancer.

Phase transformations during processing and in vitro degradation of porous calcium polyphosphates.

A 2-Step sinter/anneal treatment has been reported previously for forming porous CPP as biodegradable bone substitutes [9]. During the 2-Step annealing treatment, the heat treatment used strongly affected the rate of CPP degradation in vitro. In the present study, x-ray diffraction and (31)P solid state nuclear magnetic resonance were used to determine the phases that formed using different heat treating processes. The effect of in vitro degradation (in PBS at 37 °C, pH 7.1 or 4.5) was also studied. During CPP preparation, ß-CPP and γ-CPP were identified in powders formed from a calcium monobasic monohydrate precursor after an initial calcining treatment (10 h at 500 °C). Melting of this CPP powder (at 1100 °C), quenching and grinding formed amorphous CPP powders. Annealing powders at 585 °C (Step-1) resulted in rapid sintering to form amorphous porous CPP. Continued annealing to 650 °C resulted in crystallization to form a multi-phase structure of ß-CPP primarily plus lesser amounts of α-CPP, calcium ultra-phosphates and retained amorphous CPP. Annealing above 720 °C and up to 950 °C transformed this to ß-CPP phase. In vitro degradation of the 585 °C (Step-1 only) and 650 °C Step-2 annealed multi-phase samples occurred significantly faster than the ß-CPP samples formed by Step-2 annealing at or above 720 °C. This faster degradation was attributable to preferential degradation of thermodynamically less stable phases that formed in samples annealed at 650 °C (i.e. α-phase, ultra-phosphate and amorphous CPP). Degradation in lower pH solutions significantly increased degradation rates of the 585 and 650 °C annealed samples but had no significant effect on the ß-CPP samples.

Effect of Potassium Citrate on Calcium Phosphate Stones in a Model of Hypercalciuria.

Potassium citrate is prescribed to decrease stone recurrence in patients with calcium nephrolithiasis. Citrate binds intestinal and urine calcium and increases urine pH. Citrate, metabolized to bicarbonate, should decrease calcium excretion by reducing bone resorption and increasing renal calcium reabsorption. However, citrate binding to intestinal calcium may increase absorption and renal excretion of both phosphate and oxalate. Thus, the effect of potassium citrate on urine calcium oxalate and calcium phosphate supersaturation and stone formation is complex and difficult to predict. To study the effects of potassium citrate on urine supersaturation and stone formation, we utilized 95th-generation inbred genetic hypercalciuric stone-forming rats. Rats were fed a fixed amount of a normal calcium (1.2%) diet supplemented with potassium citrate or potassium chloride (each 4 mmol/d) for 18 weeks. Urine was collected at 6, 12, and 18 weeks. At 18 weeks, stone formation was visualized by radiography. Urine citrate, phosphate, oxalate, and pH levels were higher and urine calcium level was lower in rats fed potassium citrate. Furthermore, calcium oxalate and calcium phosphate supersaturation were higher with potassium citrate; however, uric acid supersaturation was lower. Both groups had similar numbers of exclusively calcium phosphate stones. Thus, potassium citrate effectively raises urine citrate levels and lowers urine calcium levels; however, the increases in urine pH, oxalate, and phosphate levels lead to increased calcium oxalate and calcium phosphate supersaturation. Potassium citrate induces complex changes in urine chemistries and resultant supersaturation, which may not be beneficial in preventing calcium phosphate stone formation.

Bone Marrow Stress Decreases Osteogenic Progenitors.

Age-related bone loss may be a result of declining levels of stem cells in the bone marrow. Using the Col2.3Δtk (DTK) transgenic mouse, osteoblast depletion was used as a source of marrow stress in order to investigate the effects of aging on osteogenic progenitors which reside in the marrow space. Five-month-old DTK mice were treated with one or two cycles of ganciclovir to conditionally ablate differentiated osteoblasts, whereas controls were saline-treated. Treatment cycles were two weeks in length followed by four weeks of recovery. All animals were sacrificed at 8 months of age; bone marrow stromal cells (BMSCs) were harvested for cell culture and whole bones were excised for bone quality assessment. Colony-forming unit (CFU) assays were conducted to investigate the osteogenic potential of BMSC in vitro, and RNA was extracted to assess the expression of osteoblastic genes. Bone quality assessments included bone histomorphometry, TRAP staining, microcomputed tomography, and biomechanical testing. Osteoblast depletion decreased CFU-F (fibroblast), CFU-ALP (alkaline phosphatase), and CFU-VK (von Kossa) counts and BMSC osteogenic capacity in cell culture. Ex vivo, there were no differences in bone mineral density of vertebrae or femurs between treatment groups. Histology showed a decrease in bone volume and bone connectivity with repeated osteoblast depletion; however, this was accompanied by an increase in bone formation rate. There were no notable differences in osteoclast parameters or observed bone marrow adiposity. We have developed a model that uses bone marrow stress to mimic age-related decrease in osteogenic progenitors. Our data suggest that the number of healthy BMSCs and their osteogenic potential decline with repeated osteoblast depletion. However, activity of the remaining osteoblasts increases to compensate for this loss in progenitor osteogenic potential.

1,25(OH)2D3 induces a mineralization defect and loss of bone mineral density in genetic hypercalciuric stone-forming rats.

Genetic hypercalciuric stone-forming (GHS) rats, bred to maximize urine (u) calcium (Ca) excretion, demonstrate increased intestinal Ca absorption, increased bone Ca resorption, and reduced renal Ca reabsorption, all leading to elevated uCa compared to the parental Sprague-Dawley (SD) rats. GHS rats have increased numbers of vitamin D receptors (VDRs) at each site, with normal levels of 1,25(OH)2D3 (1,25D), suggesting their VDR is undersaturated with 1,25D. We have shown that 1,25D induces a greater increase in uCa in GHS than SD rats. To examine the effect of the increased VDR on the osseous response to 1,25D, we fed GHS and SD rats an ample Ca diet and injected either 1,25D [low dose (LD) 12.5 or high dose (HD) 25 ng/100 g body weight/day] or vehicle (veh) daily for 16 days. Femoral areal bone mineral density (aBMD, by DEXA) was decreased in GHS+LD and GHS+HD relative to GHS+veh, while there was no effect on SD. Vertebral aBMD was lower in GHS compared to SD and further decreased in GHS+HD. Both femoral and L6 vertebral volumetric BMD (by µCT) were lower in GHS and further reduced by HD. Histomorphometry indicated a decreased osteoclast number in GHS+HD compared to GHS+veh or SD+HD. In tibiae, GHS+HD trabecular thickness and number increased, with a 12-fold increase in osteoid volume but only a threefold increase in bone volume. Bone formation rate was decreased in GHS+HD relative to GHS+veh, confirming the mineralization defect. The loss of BMD and the mineralization defect in GHS rats contribute to increased hypercalciuria; if these effects persist, they would result in decreased bone strength, making these bones more fracture-prone. The enhanced effect of 1,25D in GHS rats indicates that the increased VDRs are biologically active.

Collagen modifications in postmenopausal osteoporosis: advanced glycation endproducts may affect bone volume, structure and quality.

The classic model of postmenopausal osteoporosis (PM-OP) starts with the depletion of estrogen, which in turn stimulates imbalanced bone remodeling, resulting in loss of bone mass/volume. Clinically, this leads to fractures because of structural weakness. Recent work has begun to provide a more complete picture of the mechanisms of PM-OP involving oxidative stress and collagen modifications known as advanced glycation endproducts (AGEs). On one hand, AGEs may drive imbalanced bone remodeling through signaling mediated by the receptor for AGEs (RAGE), stimulating resorption and inhibiting formation. On the other hand, AGEs are associated with degraded bone material quality. Oxidative stress promotes the formation of AGEs, inhibits normal enzymatically derived crosslinking and can degrade collagen structure, thereby reducing fracture resistance. Notably, there are multiple positive feedback loops that can exacerbate the mechanisms of PM-OP associated with oxidative stress and AGEs. Anti-oxidant therapies may have the potential to inhibit the oxidative stress based mechanisms of this disease.