A novel approach to evaluate the effect of medicaments used in endodontic regeneration on root canal surface indentation.

OBJECTIVES: The aim of this study was to investigate the capability of a novel reference point indentation apparatus to test the indentation properties of root canal surface dentine treated with three intracanal medicaments used in endodontic regeneration. MATERIALS AND METHODS: Immature human premolars were selected (n = 22). Four specimens were obtained from each root and randomly assigned to three treatment groups and a control group. Each specimen was exposed to one of the three treatment pastes (triple antibiotic (TAP), double antibiotic (DAP), or calcium hydroxide (Ca(OH)2)) or neutral deionized water (control) for 1 or 4 weeks. After each time interval, the indentation properties of the root canal dentine surfaces were measured using a BioDent reference point indenter. Two-way ANOVA and Fisher's protected least significant differences were used for statistical analyses. RESULTS: Significant differences in indentation parameters and estimated hardness between all groups at both time points were found. TAP-treated dentine had the highest significant indentation parameters, followed by DAP-treated dentine, untreated control dentine, and Ca(OH)2-treated dentine, respectively. Furthermore, TAP-treated dentine had the lowest significant estimated hardness, followed by DAP-treated dentine, untreated control dentine, and Ca(OH)2-treated dentine, respectively. CONCLUSION: BioDent reference point indenter was able to detect significant differences in indentation properties of root canal dentine treated with various medicaments. CLINICAL RELEVANCE: The use of a reference point indenter is a promising approach to characterize the indentation properties of root canal surfaces without any surface modification. This might provide an in vitro mechanical measurement that is more representative of the actual clinical situation.

Multimodal epigenetic sequencing analysis (MESA) of cell-free DNA for non-invasive colorectal cancer detection.

BACKGROUND: Detecting human cancers through cell-free DNA (cfDNA) in blood is a sensitive and non-invasive option. However, capturing multiple forms of epigenetic information remains a technical and financial challenge. METHODS: To address this, we developed multimodal epigenetic sequencing analysis (MESA), a flexible and sensitive approach to capturing and integrating a diverse range of epigenetic features in cfDNA using a single experimental assay, i.e., non-disruptive bisulfite-free methylation sequencing, such as Enzymatic Methyl-seq. MESA enables simultaneous inference of four epigenetic modalities: cfDNA methylation, nucleosome occupancy, nucleosome fuzziness, and windowed protection score for regions surrounding gene promoters and polyadenylation sites. RESULTS: When applied to 690 cfDNA samples from 3 colorectal cancer clinical cohorts, MESA's novel modalities, which include nucleosome fuzziness, and genomic features, including polyadenylation sites, improve cancer detection beyond the traditional epigenetic markers of promoter DNA methylation. CONCLUSIONS: Together, MESA stands as a major advancement in the field by utilizing comprehensive and complementary epigenetic profiles of cfDNA for effective non-invasive cancer detection.

Resolution of inflammation induces osteoblast function and regulates the Wnt signaling pathway.

OBJECTIVE: Inflammation in the bone microenvironment stimulates osteoclast differentiation, resulting in uncoupling of resorption and formation. Mechanisms contributing to the inhibition of osteoblast function in inflammatory diseases, however, have not been elucidated. Rheumatoid arthritis (RA) is a prototype of an inflammatory arthritis that results in focal loss of articular bone. The paucity of bone repair in inflammatory diseases such as RA raises compelling questions regarding the impact of inflammation on bone formation. The aim of this study was to establish the mechanisms by which inflammation regulates osteoblast activity. METHODS: We characterized an innovative variant of a murine model of arthritis in which inflammation is induced in C57BL/6J mice by transfer of arthritogenic K/BxN serum and allowed to resolve. RESULTS: In the setting of resolving inflammation, bone resorption ceased and appositional osteoblast-mediated bone formation was induced, resulting in repair of eroded bone. Resolution of inflammation was accompanied by striking changes in the expression of regulators of the Wnt/ß-catenin pathway, which is critical for osteoblast differentiation and function. Down-regulation of the Wnt antagonists secreted frizzled-related protein 1 (sFRP1) and sFRP2 during the resolution phase paralleled induction of the anabolic and pro-matrix mineralization factors Wnt10b and DKK2, demonstrating the role of inflammation in regulating Wnt signaling. CONCLUSION: Repair of articular bone erosion occurs in the setting of resolving inflammation, accompanied by alterations in the Wnt signaling pathway. These data imply that in inflammatory diseases that result in persistent articular bone loss, strict control of inflammation may not be achieved and may be essential for the generation of an anabolic microenvironment that supports bone formation and repair.

An interaction between L-prostaglandin D synthase and arrestin increases PGD2 production.

L-type prostaglandin synthase (L-PGDS) produces PGD(2), a lipid mediator involved in neuromodulation and inflammation. Here, we show that L-PGDS and arrestin-3 (Arr3) interact directly and can be co-immunoprecipitated endogenously from MG-63 osteoblasts. Perinuclear L-PGDS/Arr3 co-localization is observed in PGD(2)-producing MG-63 cells and is induced by the addition of the L-PGDS substrate or co-expression of COX-2 in HEK293 cells. Inhibition of L-PGDS activity in MG-63 cells triggers redistribution of Arr3 and L-PGDS to the cytoplasm. Perinuclear localization of L-PGDS is detected in wild-type mouse embryonic fibroblasts (MEFs) but is more diffused in MEFs-arr-2(-/-)-arr-3(-/-). Arrestin-3 promotes PGD(2) production by L-PGDS in vitro. IL-1ß-induced PGD(2) production is significantly lower in MEFs-arr-2(-/-)-arr-3(-/-) than in wild-type MEFs but can be rescued by expressing Arr2 or Arr3. A peptide corresponding to amino acids 86-100 of arrestin-3 derived from its L-PGDS binding domain stimulates L-PGDS-mediated PGD(2) production in vitro and in MG-63 cells. We report the first characterization of an interactor/modulator of a PGD(2) synthase and the identification of a new function for arrestin, which may open new opportunities for improving therapies for the treatment of inflammatory diseases.

Bisphosphonate binding affinity affects drug distribution in both intracortical and trabecular bone of rabbits.

Differences in the binding affinities of bisphosphonates for bone mineral have been proposed to determine their localizations and duration of action within bone. The main objective of this study was to test the hypothesis that mineral binding affinity affects bisphosphonate distribution at the basic multicellular unit (BMU) level within both cortical and cancellous bone. To accomplish this objective, skeletally mature female rabbits (n = 8) were injected simultaneously with both low- and high-affinity bisphosphonate analogs bound to different fluorophores. Skeletal distribution was assessed in the rib, tibia, and vertebra using confocal microscopy. The staining intensity ratio between osteocytes contained within the cement line of newly formed rib osteons or within the reversal line of hemiosteons in vertebral trabeculae compared to osteocytes outside the cement/reversal line was greater for the high-affinity compared to the low-affinity compound. This indicates that the low-affinity compound distributes more equally across the cement/reversal line compared to a high-affinity compound, which concentrates mostly near surfaces. These data, from an animal model that undergoes intracortical remodeling similar to humans, demonstrate that the affinity of bisphosphonates for the bone determines the reach of the drugs in both cortical and cancellous bone.

Effects of Dietary Protein Source and Quantity on Bone Morphology and Body Composition Following a High-Protein Weight-Loss Diet in a Rat Model for Postmenopausal Obesity.

Higher protein (>30% of total energy, HP)-energy restriction (HP-ER) diets are an effective means to improve body composition and metabolic health. However, weight loss (WL) is associated with bone loss, and the impact of HP-ER diets on bone is mixed and controversial. Recent evidence suggests conflicting outcomes may stem from differences in age, hormonal status, and the predominant source of dietary protein consumed. Therefore, this study investigated the effect of four 12-week energy restriction (ER) diets varying in predominate protein source (beef, milk, soy, casein) and protein quantity (normal protein, NP 15% vs. high, 35%) on bone and body composition outcomes in 32-week-old obese, ovariectomized female rats. Overall, ER decreased body weight, bone quantity (aBMD, aBMC), bone microarchitecture, and body composition parameters. WL was greater with the NP vs. HP-beef and HP-soy diets, and muscle area decreased only with the NP diet. The HP-beef diet exacerbated WL-induced bone loss (increased trabecular separation and endocortical bone formation rates, lower bone retention and trabecular BMC, and more rod-like trabeculae) compared to the HP-soy diet. The HP-milk diet did not augment WL-induced bone loss. Results suggest that specific protein source recommendations may be needed to attenuate the adverse alterations in bone quality following an HP-ER diet in a model of postmenopausal obesity.

A multi-analyte cell-free DNA-based blood test for early detection of hepatocellular carcinoma.

The limited performance of guideline-recommended abdominal ultrasound and serum alpha-fetoprotein (AFP) highlights the urgent, unmet need for new biomarkers for more accurate detection of early hepatocellular carcinoma (HCC). To this end, we have conducted a prospective clinical validation study to evaluate the performance of the HelioLiver Test, a multi-analyte blood test combining cell-free DNA methylation patterns, clinical variables, and protein tumor markers. A blinded, multicenter validation study was performed with 247 subjects, including 122 subjects with HCC and 125 control subjects with chronic liver disease. The performance of the HelioLiver Test was compared with AFP and the GALAD score as established HCC surveillance blood tests. The performance of the HelioLiver Test (area under the receiver operating characteristic curve [AUROC] = 0.944) was superior to both AFP (AUROC = 0.851; p < 0.0001) and GALAD (AUROC = 0.899; p < 0.0001). Using a prespecified diagnostic algorithm, the HelioLiver Test showed sensitivities of 85% (95% confidence interval [CI], 78%-90%) for HCC of any stage and 76% (95% CI, 60%-87%) for early stage (American Joint Committee on Cancer [AJCC] I and II) HCC. In contrast, AFP (≥20 ng/mL) alone and the GALAD score (≥-0.63) showed lower sensitivities of 62% (95% CI, 54%-70%) and 75% (95% CI, 67%-82%) for HCC overall, and 57% (95% CI, 40%-71%) and 65% (95% CI, 49%-79%) for early stage (AJCC I and II) HCC, respectively. The specificities of the HelioLiver Test (91%; 95% CI, 85%-95%), AFP (97%; 95% CI, 92%-99%), and the GALAD score (94%; 95% CI, 88%-97%) were similar for control subjects. The HelioLiver Test showed superior performance for HCC detection compared to with both AFP and the GALAD score and warrants further evaluation in HCC surveillance settings.

ANKRD13C acts as a molecular chaperone for G protein-coupled receptors.

Although the mechanisms that regulate folding and maturation of newly synthesized G protein-coupled receptors are crucial for their function, they remain poorly characterized. By yeast two-hybrid screening, we have isolated ANKRD13C, a protein of unknown function, as an interacting partner for the DP receptor for prostaglandin D(2). In the present study we report the characterization of this novel protein as a regulator of DP biogenesis and trafficking in the biosynthetic pathway. Co-localization by confocal microscopy with an endoplasmic reticulum (ER) marker, subcellular fractionation experiments, and demonstration of the interaction between ANKRD13C and the cytoplasmic C terminus of DP suggest that ANKRD13C is a protein associated with the cytosolic side of ER membranes. Co-expression of ANKRD13C with DP initially increased receptor protein levels, whereas siRNA-mediated knockdown of endogenous ANKRD13C decreased them. Pulse-chase experiments indicated that ANKRD13C can promote the biogenesis of DP by inhibiting the degradation of newly synthesized receptors. However, a prolonged interaction between ANKRD13C and DP resulted in ER retention of misfolded/unassembled forms of the receptor and to their proteasome-mediated degradation. ANKRD13C also regulated the expression of other GPCRs tested (CRTH2, thromboxane A(2) (TPα), and ß2-adrenergic receptor), whereas it did not affect the expression of green fluorescent protein, GRK2 (G protein-coupled receptor kinase 2), and VSVG (vesicular stomatitis virus glycoprotein), showing specificity toward G protein-coupled receptors. Altogether, these results suggest that ANKRD13C acts as a molecular chaperone for G protein-coupled receptors, regulating their biogenesis and exit from the ER.

Differential regulation of the signaling and trafficking of the two prostaglandin D2 receptors, prostanoid DP receptor and CRTH2.

Prostaglandin D2 (PGD2) exerts its actions on two G protein-coupled receptors, the prostanoid DP receptor and CRTH2 (chemoattractant homologous receptor expressed on TH2 cells). Here, we characterize the regulation of the signaling and trafficking of the prostanoid DP receptor and CRTH2. Time-course and dose-response curves showed that both receptors expressed in HEK293 cells internalized maximally after 2 h of stimulation with 1 microM PGD2. Co-expression of the G protein-coupled receptor kinases GRK2, GRK5 or GRK6 increased agonist-induced internalization of CRTH2, while only GRK2 had an effect on the internalization of the prostanoid DP receptor. Protein kinase C (PKC) activation stimulated the internalization of both receptors. Interestingly, only PGD2-induced internalization of CRTH2, and not of prostanoid DP receptor, was decreased by inhibition of PKC or protein kinase A (PKA). Our data also indicate that CRTH2 is subjected to basal phosphorylation by PKA, which appears to be involved in CRTH2 internalization. Prostanoid DP receptor internalization was promoted by co-expression of arrestin-2 and -3, while the internalization of CRTH2 was increased by co-expression of arrestin-3 only. The detection of prostanoid DP receptor and CRTH2 internalization was reduced by the co-expression of Rab4 and Rab11, respectively, suggesting differential regulation of receptor recycling. Moreover, immunofluorescence microscopy experiments showed that the prostanoid DP receptor specifically co-localized with Rab4, and CRTH2 with Rab11. The signaling of the prostanoid DP receptor was regulated by GRK2 overexpression, while that of CRTH2 was modulated by overexpression of GRK2, -5 and -6. Our results show a differential regulation of the prostanoid DP receptor and CRTH2, two receptors for PGD2.

Oligomerization of the alpha and beta isoforms of the thromboxane A2 receptor: relevance to receptor signaling and endocytosis.

Thromboxane A(2) (TXA(2)) is a potent mediator of inflammation, vasoconstriction and oxidative stress. The TXA(2) receptor (TP) is a G protein-coupled receptor (GPCR) that is expressed as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues) that share the first 328 residues. For many years GPCRs were assumed to exist and function as monomeric species, but increasing evidence suggests that a dimer is the minimal functional unit of GPCRs. In the present report, using co-immunoprecipitation of differentially tagged TP expressed in HEK293 cells, we demonstrate that TPalpha and TPbeta form homo- and hetero-oligomers. Immunoblotting of lysates from human platelets with an anti-TP specific antibody revealed the presence of endogenously expressed TP oligomers. We show that TP oligomerization is an agonist-independent process highly affected by the reducing agent dithiothreitol suggesting the involvement of disulfide bonds in TP oligomerization. Over-expression of G protein-coupled receptor kinases and arrestins did not modulate the extent of receptor dimerization/oligomerization. Co-expression of two TP signaling-deficient mutants, R60L and E2402R, resulted in rescuing of receptor signal transduction suggesting that dimers/oligomers constitute the functional units of this receptor. Interestingly, TPalpha which does not undergo constitutive or agonist-induced endocytosis on its own was subjected to both types of endocytosis when co-expressed with TPbeta, indicating that TPalpha can display intracellular trafficking when complexed through hetero-oligomerization with TPbeta.

Production of prostaglandin D(2) by human osteoblasts and modulation of osteoprotegerin, RANKL, and cellular migration by DP and CRTH2 receptors.

UNLABELLED: Human osteoblasts produce PGD(2), which acts on the DP receptor to decrease osteoprotegerin production and on the CRTH2 receptor to decrease RANKL expression and to induce osteoblast chemotaxis. These results indicate that activation of CRTH2 may lead to an anabolic response in bone. INTRODUCTION: Whereas the actions of prostaglandin (PG)E(2) as a modulator of bone and osteoblast function are relatively well characterized, little is known about PGD(2) and bone metabolism. The objectives of this study were to determine if human osteoblasts can produce PGD(2), which prostaglandin D(2) synthases are implicated in this synthesis, to identify the PGD(2) receptors (DP and CRTH2) on these cells and to characterize the biological effects resulting from their activation. MATERIALS AND METHODS: RT-PCR analysis and immunohistochemistry were used to detect PGD(2) receptor and synthases in cultured human osteoblasts. Immunohistochemistry was used to identify the synthases and receptors in human bone tissue. Intracellular cAMP and calcium levels were determined to verify receptor activation. The cells were stimulated with PGD(2) or the specific agonists BW 245C (DP) and DK-PGD(2) (CRTH2), and the resulting effects on osteoprotegerin (OPG) secretion, RANKL expression, and chemotaxis were determined. Osteoblast production of PGD(2) was evaluated by measuring PGD(2) in the culture supernatants after stimulation with interleukin (IL)-1, TNF-alpha, PTH, vascular endothelial growth factor (VEGF), and insulin-like growth factor I (IGF-I). RESULTS: Human osteoblasts in culture generated PGD(2) when stimulated. Both osteoblasts in culture and in situ present the lipocalin-type PGD(2) synthase only. Both DP and CRTH2 receptors were present in human osteoblasts in culture and in situ. Stimulation of DP resulted in an increase in cAMP, whereas CRTH2 increased the intracellular calcium level. OPG production was reduced by 60% after DP receptor stimulation, whereas CRTH2 receptor stimulation decreased RANKL expression on human osteoblasts. As reported for other cell types, CRTH2 was a potent inducer of chemotaxis for human osteoblasts in culture. CONCLUSIONS: Human osteoblasts in culture produce PGD(2) under biologically relevant stimuli through the lipocalin-type PGD(2) synthase (L-PGDS) pathway. As an autacoid, PGD(2) can act on DP and CRTH2 receptors, both present on these cells. Specific activation of CRTH2 could lead directly and indirectly to an anabolic response in bone.

Role of extracellular cysteine residues in dimerization/oligomerization of the human prostacyclin receptor.

Prostacyclin activation of prostanoid IP receptors may result in pain sensation, inflammatory responses, inhibition of platelet aggregation, and vasodilation in vascular tissue. The prostanoid IP receptor is a G-protein-coupled receptor. In the present study, we investigated the determinants responsible, at least in part, for the prostacyclin receptor (IP) dimerization/oligomerization. Using co-immunoprecipitation of differentially tagged IP expressed in COS-7 cells, we demonstrate that IP can form dimers and oligomers. Treatment of IP-expressing cells with the stable agonist carbaprostacyclin failed to alter the ratios of oligomeric/dimeric/monomeric forms of the receptor, suggesting that IP dimerization/oligomerization is an agonist-independent process. The reducing agents dithiothreitol and 2-mercaptoethanol were highly efficient in converting the receptor from its oligomeric form to the monomeric state, indicating the involvement of disulfide bonds in IP oligomerization. Immunoblotting of the osteoblastic MG-63 cell line lysates with an anti-IP specific antibody revealed the presence of endogenous IP oligomers which were converted to dimers and monomers upon treatment with dithiothreitol. Individual substitutions of the four extracellular IP Cys residues (Cys(5), Cys(92), Cys(165) and Cys(170)) for Ser resulted in greatly decreased receptor protein expression in COS-7 cells. The C92-170S double mutant showed receptor protein expression level similar to the individual mutants. However, expression of the C92-165S and C165-170S mutants was drastically reduced, suggesting that there was formation of disulfide bonds between Cys(5) and Cys(165), and between Cys(92) and Cys(170). The Cys receptor mutants showed altered oligomer/dimer/monomer ratios. Dimerization/oligomerization likely occurs intracellularly since these Cys receptor mutants could still form dimers/oligomers despite their lack of expression at the cell surface.

Nanoscale changes in collagen are reflected in physical and mechanical properties of bone at the microscale in diabetic rats.

Diabetes detrimentally affects the musculoskeletal system by stiffening the collagen matrix due to increased advanced glycation end products (AGEs). In this study, tibiae and tendon from Zucker diabetic Sprague-Dawley (ZDSD) rats were compared to Sprague-Dawley derived controls (CD) using Atomic Force Microscopy. ZDSD and CD tibiae were compared using Raman Spectroscopy and Reference Point Indentation (RPI). ZDSD bone had a significantly different distribution of collagen D-spacing than CD (p=0.015; ZDSD n=294 fibrils; CD n=274 fibrils) which was more variable and shifted to higher values. This shift between ZDSD and CD D-spacing distribution was more pronounced in tendon (p<0.001; ZDSD n=350; CD n=371). Raman revealed significant increases in measures of bone matrix mineralization in ZDSD (PO4(3-) ν1/Amide I p=0.008; PO4(3-) ν1/CH2 wag p=0.047; n=5 per group) despite lower bone mineral density (aBMD) and ash fraction indicating diabetes may preferentially reduce the Raman signature of collagen. Decreased indentation distance increase (p=0.010) and creep indentation distance (p=0.040) measured by RPI (n=9 per group) in ZDSD rats suggest a matrix more resistant to indentation under the high stresses associated with RPI at this length scale. There were significant correlations between Raman and RPI measurements in the ZDSD population (n=18 locations) but not the CD population (n=16 locations) indicating that while RPI is relatively unaffected by biological noise, it is sensitive to disease-induced compositional changes. In conclusion, diabetes in the ZDSD rat causes changes to the nanoscale morphology of collagen that result in compositional and mechanical effects in bone at the microscale.

Multiscale analysis of morphology and mechanics in tail tendon from the ZDSD rat model of type 2 diabetes.

Type 2 diabetes (T2D) impacts multiple organ systems including the circulatory, renal, nervous and musculoskeletal systems. In collagen-based tissues, one mechanism that may be responsible for detrimental mechanical impacts of T2D is the formation of advanced glycation end products (AGEs) leading to increased collagen stiffness and decreased toughness, resulting in brittle tissue behavior. The purpose of this study was to investigate tendon mechanical properties from normal and diabetic rats at two distinct length scales, testing the hypothesis that increased stiffness and strength and decreased toughness at the fiber level would be associated with alterations in nanoscale morphology and mechanics. Individual fascicles from female Zucker diabetic Sprague-Dawley (ZDSD) rats had no differences in fascicle-level mechanical properties but had increased material-level strength and stiffness versus control rats (CD). At the nanoscale, collagen fibril D-spacing was shifted towards higher spacing values in diabetic ZDSD fibrils. The distribution of nanoscale modulus values was also shifted to higher values. Material-level strength and stiffness from whole fiber tests were increased in ZDSD tails. Correlations between nanoscale and microscale properties indicate a direct positive relationship between the two length scales, most notably in the relationship between nanoscale and microscale modulus. These findings indicate that diabetes-induced changes in material strength and modulus were driven by alterations at the nanoscale.

Raloxifene prevents skeletal fragility in adult female Zucker Diabetic Sprague-Dawley rats.

Fracture risk in type 2 diabetes is increased despite normal or high bone mineral density, implicating poor bone quality as a risk factor. Raloxifene improves bone material and mechanical properties independent of bone mineral density. This study aimed to determine if raloxifene prevents the negative effects of diabetes on skeletal fragility in diabetes-prone rats. Adult Zucker Diabetic Sprague-Dawley (ZDSD) female rats (20-week-old, n = 24) were fed a diabetogenic high-fat diet and were randomized to receive daily subcutaneous injections of raloxifene or vehicle for 12 weeks. Blood glucose was measured weekly and glycated hemoglobin was measured at baseline and 12 weeks. At sacrifice, femora and lumbar vertebrae were harvested for imaging and mechanical testing. Raloxifene-treated rats had a lower incidence of type 2 diabetes compared with vehicle-treated rats. In addition, raloxifene-treated rats had blood glucose levels significantly lower than both diabetic vehicle-treated rats as well as vehicle-treated rats that did not become diabetic. Femoral toughness was greater in raloxifene-treated rats compared with both diabetic and non-diabetic vehicle-treated ZDSD rats, due to greater energy absorption in the post-yield region of the stress-strain curve. Similar differences between groups were observed for the structural (extrinsic) mechanical properties of energy-to-failure, post-yield energy-to-failure, and post-yield displacement. These results show that raloxifene is beneficial in preventing the onset of diabetes and improving bone material properties in the diabetes-prone ZDSD rat. This presents unique therapeutic potential for raloxifene in preserving bone quality in diabetes as well as in diabetes prevention, if these results can be supported by future experimental and clinical studies.

Bone cell-independent benefits of raloxifene on the skeleton: a novel mechanism for improving bone material properties.

Raloxifene is an FDA approved agent used to treat bone loss and decrease fracture risk. In clinical trials and animal studies, raloxifene reduces fracture risk and improves bone mechanical properties, but the mechanisms of action remain unclear because these benefits occur largely independent of changes to bone mass. Using a novel experimental approach, machined bone beams, both from mature male canine and human male donors, were depleted of living cells and then exposed to raloxifene ex vivo. Our data show that ex vivo exposure of non-viable bone to raloxifene improves intrinsic toughness, both in canine and human cortical bone beams tested by 4-point bending. These effects are cell-independent and appear to be mediated by an increase in matrix bound water, assessed using basic gravimetric weighing and sophisticated ultrashort echo time magnetic resonance imaging. The hydroxyl groups (OH) on raloxifene were shown to be important in both the water and toughness increases. Wide and small angle X-ray scattering patterns during 4-pt bending show that raloxifene alters the transfer of load between the collagen matrix and the mineral crystals, placing lower strains on the mineral, and allowing greater overall deformation prior to failure. Collectively, these findings provide a possible mechanistic explanation for the therapeutic effect of raloxifene and more importantly identify a cell-independent mechanism that can be utilized for novel pharmacological approaches for enhancing bone strength.

Reference-point indentation correlates with bone toughness assessed using whole-bone traditional mechanical testing.

Traditional bone mechanical testing techniques require excised bone and destructive sample preparation. Recently, a cyclic-microindentation technique, reference-point indentation (RPI), was described that allows bone to be tested in a clinical setting, permitting the analysis of changes to bone material properties over time. Because this is a new technique, it has not been clear how the measurements generated by RPI are related to the material properties of bone measured by standard techniques. In this paper, we describe our experience with the RPI technique, and correlate the results obtained by RPI with those of traditional mechanical testing, namely 3-point bending and axial compression. Using different animal models, we report that apparent bone material toughness obtained from 3-point bending and axial compression is inversely correlated with the indentation distance increase (IDI) obtained from RPI with r(2) values ranging from 0.50 to 0.57. We also show that conditions or treatments previously shown to cause differences in toughness, including diabetes and bisphosphonate treatment, had significantly different IDI values compared to controls. Collectively these results provide a starting point for understanding how RPI relates to traditional mechanical testing results.

Elevated mechanical loading when young provides lifelong benefits to cortical bone properties in female rats independent of a surgically induced menopause.

Exercise that mechanically loads the skeleton is advocated when young to enhance lifelong bone health. Whether the skeletal benefits of elevated loading when young persist into adulthood and after menopause are important questions. This study investigated the influence of a surgically induced menopause in female Sprague-Dawley rats on the lifelong maintenance of the cortical bone benefits of skeletal loading when young. Animals had their right forearm extrinsically loaded 3 d/wk between 4 and 10 weeks of age using the forearm axial compression loading model. Left forearms were internal controls and not loaded. Animals were subsequently detrained (restricted to cage activities) for 94 weeks (until age 2 years), with ovariectomy (OVX) or sham-OVX surgery being performed at 24 weeks of age. Loading enhanced midshaft ulna cortical bone mass, structure, and estimated strength. These benefits persisted lifelong and contributed to loaded ulnas having greater strength after detraining. Loading also had effects on cortical bone quality. The benefits of loading when young were not influenced by a surgically induced menopause because there were no interactions between loading and surgery. However, OVX had independent effects on cortical bone mass, structure, and estimated strength at early postsurgery time points (up to age 58 weeks) and bone quality measures. These data indicate skeletal loading when young had lifelong benefits on cortical bone properties that persisted independent of a surgically induced menopause. This suggests that skeletal loading associated with exercise when young may provide lifelong antifracture benefits by priming the skeleton to offset the cortical bone changes associated with aging and menopause.

In vivo reference point indentation reveals positive effects of raloxifene on mechanical properties following 6 months of treatment in skeletally mature beagle dogs.

Raloxifene treatment has been shown previously to positively affect bone mechanical properties following 1 year of treatment in skeletally mature dogs. Reference point indentation (RPI) can be used for in vivo assessment of mechanical properties and has been shown to produce values that are highly correlated with properties derived from traditional mechanical testing. The goal of this study was to use RPI to determine if raloxifene-induced alterations in mechanical properties occurred after 6 months of treatment. Twelve skeletally mature female beagle dogs were treated for 6 months with oral doses of saline vehicle (VEH, 1 ml/kg/day) or a clinically relevant dose of raloxifene (RAL, 0.5 mg/kg/day). At 6 months, all animals underwent in vivo RPI (10N force, 10 cycles) of the anterior tibial midshaft. RPI data were analyzed using a custom MATLAB program, designed to provide cycle-by-cycle data from the RPI test and validated against the manufacturer-provided software. Indentation distance increase (IDI), a parameter that is inversely related to bone toughness, was significantly lower in RAL-treated animals compared to VEH (-16.5%), suggesting increased bone toughness. Energy absorption within the first cycle was significantly lower with RAL compared to VEH (-21%). These data build on previous work that has documented positive effects of raloxifene on material properties by showing that these changes exist after 6 months.

Inverse agonist and pharmacochaperone properties of MK-0524 on the prostanoid DP1 receptor.

Prostaglandin D2 (PGD2) acts through two G protein-coupled receptors (GPCRs), the prostanoid DP receptor and CRTH2 also known as DP1 and DP2, respectively. Several previously characterized GPCR antagonists are now classified as inverse agonists and a number of GPCR ligands are known to display pharmacochaperone activity towards a given receptor. Here, we demonstrate that a DP1 specific antagonist, MK-0524 (also known as laropiprant), decreased basal levels of intracellular cAMP produced by DP1, a Gα(s)-coupled receptor, in HEK293 cells. This reduction in cAMP levels was not altered by pertussis toxin treatment, indicating that MK-0524 did not induce coupling of DP1 to Gα(i/o) proteins and that this ligand is a DP1 inverse agonist. Basal ERK1/2 activation by DP1 was not modulated by MK-0524. Interestingly, treatment of HEK293 cells expressing Flag-tagged DP1 with MK-0524 promoted DP1 cell surface expression time-dependently to reach a maximum increase of 50% compared to control after 24 h. In contrast, PGD2 induced the internalization of 75% of cell surface DP1 after the same time of stimulation. The increase in DP1 cell surface targeting by MK-0524 was inhibited by Brefeldin A, an inhibitor of transport from the endoplasmic reticulum-Golgi to the plasma membrane. Confocal microscopy confirmed that a large population of DP1 remained trapped intracellularly and co-localized with calnexin, an endoplasmic reticulum marker. Redistribution of DP1 from intracellular compartments to the plasma membrane was observed following treatment with MK-0524 for 24 h. Furthermore, MK-0524 promoted the interaction between DP1 and the ANKRD13C protein, which we showed previously to display chaperone-like effects towards the receptor. We thus report that MK-0524 is an inverse agonist and a pharmacochaperone of DP1. Our findings may have important implications during therapeutic treatments with MK-0524 and for the development of new molecules targeting DP1.