Calcium Content Quantification Research Articles

Repression of the antiporter SLC7A11/glutathione/glutathione peroxidase 4 axis drives ferroptosis of vascular smooth muscle cells to facilitate vascular calcification

Vascular calcification is a common pathologic condition in patients with chronic kidney disease (CKD). Cell death such as apoptosis plays a critical role in vascular calcification. Ferroptosis is a type of iron-catalyzed and regulated cell death resulting from excessive iron-dependent reactive oxygen species and lipid peroxidation. However, it is unclear whether ferroptosis of vascular smooth muscle cells (VSMCs) regulates vascular calcification in CKD. Our results showed that high calcium and phosphate concentrations induced ferroptosis in rat VSMCs invitro. Inhibition of ferroptosis by ferrostatin-1 dose-dependently reduced mineral deposition in rat VSMCs under pro-osteogenic conditions, as indicated by alizarin red staining and quantification of calcium content. In addition, gene expression analysis revealed that ferrostatin-1 inhibited osteogenic differentiation of rat VSMCs. Similarly, ferrostatin-1 remarkably attenuated calcification of rat and human arterial rings exvivo and aortic calcification in vitamin D3-overloaded mice invivo. Moreover, inhibition of ferroptosis by either ferrostatin-1 or deferoxamine attenuated aortic calcification in rats with CKD. Mechanistically, high calcium and phosphate downregulated expression of SLC7A11 (a cystine-glutamate antiporter), and reduced glutathione (GSH) content in VSMCs. Additionally, GSH depletion induced by erastin (a small molecule initiating ferroptotic cell death) significantly promoted calcification of VSMCs under pro-osteogenic conditions, whereas GSH supplement by N-acetylcysteine reduced calcification of VSMCs. Consistently, knockdown of SLC7A11 by siRNA markedly promoted VSMC calcification. Furthermore, high calcium and phosphate downregulated glutathione peroxidase 4 (GPX4) expression, and reduced glutathione peroxidase activity. Inhibition of GPX4 by RSL3 promoted VSMC calcification. Thus, repression of the SLC7A11/GSH/GPX4 axis triggers ferroptosis of VSMCs to promote vascular calcification under CKD conditions, providing a novel targeting strategy for vascular calcification.

Spermidine inhibits vascular calcification in chronic kidney disease through modulation of SIRT1 signaling pathway.

Vascular calcification is a common pathologic condition in patients with chronic kidney disease (CKD) and aging individuals. It has been established that vascular calcification is a gene‐regulated biological process resembling osteogenesis involving osteogenic differentiation. However, there is no efficient treatment available for vascular calcification so far. The natural polyamine spermidine has been demonstrated to increase life span and protect against cardiovascular disease. It is unclear whether spermidine supplementation inhibits vascular calcification in CKD. Alizarin red staining and quantification of calcium content showed that spermidine treatment markedly reduced mineral deposition in both rat and human vascular smooth muscle cells (VSMCs) under osteogenic conditions. Additionally, western blot analysis revealed that spermidine treatment inhibited osteogenic differentiation of rat and human VSMCs. Moreover, spermidine treatment remarkably attenuated calcification of rat and human arterial rings ex vivo and aortic calcification in rats with CKD. Furthermore, treatment with spermidine induced the upregulation of Sirtuin 1 (SIRT1) in VSMCs and resulted in the downregulation of endoplasmic reticulum (ER) stress signaling components, such as activating transcription factor 4 (ATF4) and CCAAT/enhancer‐binding protein homologous protein (CHOP). Both pharmacological inhibition of SIRT1 by SIRT1 inhibitor EX527 and knockdown of SIRT1 by siRNA markedly blocked the inhibitory effect of spermidine on VSMC calcification. Consistently, EX527 abrogated the inhibitory effect of spermidine on aortic calcification in CKD rats. We for the first time demonstrate that spermidine alleviates vascular calcification in CKD by upregulating SIRT1 and inhibiting ER stress, and this may develop a promising therapeutic treatment to ameliorate vascular calcification in CKD.

Nanocomposite scaffolds composed of Apacite (apatite-calcite) nanostructures, poly (ε-caprolactone) and poly (2-hydroxyethylmethacrylate): The effect of nanostructures on physico-mechanical properties and osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro.

Nanocomposite scaffolds were fabricated from poly (ε-caprolactone) (PCL), Poly (2-hydroxyethylmethacrylate) (PHEMA), and Apacite (Apatite-calcite) nanostructures (15 and 25wt%). The nanoscale structure, physical and chemical properties, mechanical properties, hydrophilic behavior, degradability and osteogenic properties of the fabricated scaffolds were evaluated. The results showed that the mechanical strength, degradation, wetting ability, and mechanical strength of PCL-PHEMA scaffolds significantly increases upon inclusion of Apacite nanoparticles up to 25wt%. Accordingly, the best mechanical values (E~7.109MPa and σ~0.414MPa) and highest degradability (32% within 96h) were recorded for PCL-PHEMA scaffolds containing 25wt% of Apacite. Furthermore, highest porosity and roughness were observed in the PCL-PHEMA/25% Apacite as a result of the Apacite nanoparticles inclusion. There was no cytotoxicity recorded for the fabricated scaffolds based on the results obtained from MTT assay and acridine orange staining. Alkaline phosphatase activity, calcium content quantification, Van Kossa staining, FESEM and real time PCR tests confirmed the biomineralization, and the differentiation potential of the nanocomposite scaffolds. Overall, the 3D structure, optimum porosity and balanced dissolution rate of PCL-PHEMA/25% Apacite providing a balanced microenvironment resulted in improved cell adhesion, cell behavior, and replication, as well as osteogenic induction of human bone-marrow-derived mesenchymal stem cells (hBM-MSCs).

Feasibility of laser-induced breakdown spectroscopy (LIBS) as an at-line validation tool for calcium determination in infant formula

In this study, a 150 mJ laser-induced breakdown spectroscopy (LIBS) system was assessed to determine calcium content in infant formula (IF) samples. LIBS is a promising emission spectroscopic technique for elemental analysis, which offers advantages over conventional methods such as real-time analyses, little to no sample preparation and ease of use. The aim of this work was to evaluate the feasibility of LIBS as an at-line tool for IF manufacturing. To this end, IF mixtures with varying content of calcium were prepared over a range (approx. 1.5–7 mg/g of calcium) selected to be in conformity with the guidelines provided by the Codex Alimentarius Commission. Atomic Absorption Spectroscopy (AAS) was used as the reference method for mineral content determination in IF. Partial least squares regression (PLSR) was applied to the LIBS spectral data to develop a calibration model for calcium content quantification. A validation approach was then carried out to investigate the robustness of the model, which showed a good fit with values of R2cv of 0.90 for cross-validation and a R2p 0.85 for external validation. Low values for root mean square errors of cross-validation (RMSECV) and prediction (RMSEP) of 0.62 mg/g and 0.68 mg/g were obtained. Furthermore, this study also illustrated the possibility of LIBS to provide mineral prediction maps as a useful tool for testing sample homogeneity.

Catheter based inhibition of arterial calcification by bisphosphonates in an experimental atherosclerotic rabbit animal model

BackgroundVascular calcification is an active process, sharing common molecular mechanisms with bone formation. Bisphosphonates are components, which inhibit calcification. The aim of the present study was to evaluate the safety and effectiveness of local delivery of the bisphosphonate zoledronate on inhibition of calcium formation in the arterial wall in an experimental animal model. MethodsSixteen New Zealand rabbits were placed on vitamin D enriched atherogenic diet for 3weeks. Subsequently, all animals underwent angiography of abdominal aorta and common iliac arteries. A mixture containing 500μg/l zoledronate was delivered on the vascular wall of the target iliac artery, using a dedicated balloon catheter. A placebo mixture was administered on the contralateral iliac artery of each animal, which was used as control. At 28days all animals were sacrificed. Histologic sections of each common iliac artery were stained with hematoxylin–eosin and von Kossa. Computer-assisted histomorphometry was performed for the calcium content quantification of each section from the target and the control iliac artery. ResultsIn all animals the local delivery of zoledronate and placebo mixtures was successful and uncomplicated. The mean percentage of the calcium content of the media was higher in the control artery segments compared to the target (2.66±0.73 versus 1.08±0.62 % of the area of the media, p<0.01). ConclusionsInhibition of vascular calcification by local catheter-based delivery of bisphosphonate zolendronic acid is effective without evident short-term complications. These finding and its potential clinical implication remain to be confirmed in human studies.

Quantification of calcium content in bone by using ToF-SIMS–a first approach

The determination of the spatially resolved calcium distribution and concentration in bone is essential for the assessment of bone quality. It enables the diagnosis and elucidation of bone diseases, the course of bone remodelling and the assessment of bone quality at interfaces to implants. With time-of-flight secondary ion mass spectrometry (ToF-SIMS) the calcium distribution in bone cross sections is mapped semi-quantitatively with a lateral resolution of up to 1 μm. As standards for the calibration of the ToF-SIMS data calcium hydroxyapatite collagen scaffolds with different compositions were synthesized. The standards were characterised by loss of ignition, x-ray diffractometry (XRD) and x-ray photoelectron spectroscopy (XPS). The secondary ion count rate for calcium and the calcium content of the standards show a linear dependence. The obtained calibration curve is used for the quantification of the calcium content in the bone of rats. The calcium concentration within an animal model for osteoporosis induction is monitored. Exemplarily the calcium content of the bones was quantified by XPS for validation of the results. Furthermore a calcium mass image is compared with an XPS image to demonstrate the better lateral resolution of ToF-SIMS which advances the locally resolved quantification of the calcium content.

α-Lipoic acid attenuates coxsackievirus B3-induced ectopic calcification in heart, pancreas, and lung

Ectopic mineralization of soft tissues is known to be a typical response to systemic imbalance of various metabolic factors as well as tissue injury, leading to severe clinical consequences. In this study, coxsackievirus B3 (CVB3) infection in mice resulted in significant tissue injury, especially in the heart and pancreas. Inflammatory damage and apoptotic cell death were observed in CVB3-infected heart and pancreas tissues. Along with tissue damage, substantial ectopic calcification was detected in CVB3-infected heart, pancreas, and lung tissues, as determined by von Kossa staining and calcium content quantification. In addition, CVB3 infection induced upregulation of osteogenic signals, including six genes (BMP2, SPARC, Runx2, osteopontin, collagen type I, and osterix) in the heart, three genes (SPARC, osteopontin, and collagen type I) in the pancreas, and two genes (BMP2 and alkaline phosphatase) in the lung, as determined by quantitative real-time PCR analysis. Intriguingly, we showed that α-lipoic acid diminished CVB3-mediated inflammatory and apoptotic tissue damage, subsequently ameliorating ectopic calcification via the suppression of osteogenic signals. Collectively, our data provide evidence that ectopic calcification induced by CVB3 infection is implicated in the induction of osteogenic propensity, and α-lipoic acid may be a potential therapeutic agent to ameliorate pathologic calcification.

1026 EARLY BRIEF TREATMENT WITH ANGIOTENSIN CONVERTING ENZYME INHIBITOR, PERINDOPRIL, PREVENTS AORTIC CALCIFICATION IN ADULT SPONTANEOUSLY HYPERTENSIVE RATS

Background: Aortic calcification is a potent risk marker of cardiovascular events. Aortic calcification contributes to arterial stiffness and increased pulse pressure. Early angiotensin converting enzyme inhibition (ACEi) in spontaneously hypertensive rats (SHR) during a critical treatment window prevents aortic stiffness in adulthood. However, the effect of ACEi on aortic calcification is unknown. Method: ACEi (perindopril, 3 mg/kg/day, by gavage) was delivered during the pre- (6-10 weeks, SHR(Tx)y, n = 6) and established (20-24 weeks of age, SHR(Tx)o, n = ?) hypertensive phases. Vascular stiffness of the thoracic and abdominal aortic segments at their corresponding anaesthetised mean aortic pressure (MAP) was calculated from beat-to-beat pulse wave velocity (PWV) measurements in-vivo, using two 1.4F pressure catheters introduced to the thoracic and abdominal aorta, positioned a known distance apart. Aortas were harvested post-mortem and divided into thoracic and abdominal segments at the renal bifurcation for quantification of calcium content using atomic absorption spectrophotometry. Results were compared to untreated SHR, and the normotensive control strain, Wistar-Kyoto (WKY). Results: Perindopril significantly decreased MAP (-43%, P=0.0005), isobaric thoracic PWV (-19%, P=0.002) but not abdominal PWV. Thoracic aorta of the SHR(Tx)y group calcified 54% less than that of SHR controls (p=0.001), and approximated values of the normotensive control strain, with no difference detected in the abdominal aorta (p=0.09). The SHR(Tx)o group did not differ from SHR controls in any measured parameters. Conclusion: This study shows direct evidence of the persistent effect of early, but not late, ACEi in prevention of arterial stiffness associated with hypertension and corresponding aortic calcification.

A modular approach to the engineering of a centimeter-sized bone tissue construct with human amniotic mesenchymal stem cells-laden microcarriers

Tissue engineering of clinical-relevant large tissue constructs remains a big challenge due to the mass transfer limit. A modular approach via the assembling of modular tissues thus eliminating the mass transfer limit holds great promise for fabricating centimeter-sized constructs. In the present study, we investigated the feasibility of using microcarriers seeded with adult mesenchymal stem cells (MSCs) to fabricate a large bone tissue. It was demonstrated that human amniotic MSCs (hAMSCs) were efficiently seeded onto CultiSpher S microcarriers (made of porcine gelatin) in a spinner flask and quickly proliferated while retaining a great viability. Within a total culture period of 28 days, using a two-stage culture strategy, hAMSCs-laden microcarriers with a high cell density were prepared at the first stage and the cells were then directly induced to undergo osteogenic differentiation in the same culture flask. During this cultivation process, the aggregation of cell-laden microcarriers was apparent, which resulted in aggregates of 700–800 μm, a size permissive for maintaining high cell viability. The osteogenic differentiation of hAMSCs on microcarriers was confirmed with increased mineral deposition (Alizarin red S staining and quantification of calcium content), ALP activity as well as gene expression of osteogenic markers (collagen type I and osteocalcin). These modular bone-like tissues were used as building blocks to fabricate a macroscopic bone construct in a cylindrical perfusion culture chamber (2 cm in diameter). After a 7-day perfusion culture, these modular tissues readily assembled into a centimeter-sized construct (diameter × height: 2 cm × 1 cm). Both good cell viability and fairly homogenous distribution of cellular content and bone-characteristic ECM within the macrotissue were elaborated. This paper provided a proof-of-concept study for modularly engineering clinical-relevant large tissue replacements with cell-laden microcarriers.

マルチディテクタＣＴにおけるハイブリッド型逐次近似画像再構成（ｉＤｏｓｅ）アルゴリズムを用いた低線量撮像時の冠動脈石灰化の定量評価：フィルタ補正逆投影法との比較

To evaluate the usefulness of hybrid iterative reconstruction (iDose) for quantification of calcium content in the coronary artery on 64-detector computed tomography (CT), an anthropomorphic cardiac CT phantom containing cylinders with known calcium content was scanned at tube current-time products of 15, 20, 25, and 50 mAs using 64-detector CT. The images obtained at 15, 20, 25, and 50 mAs were reconstructed using filtered back projection (FBP), and those at 15, 20, and 25 mAs were also reconstructed using iDose. Then the volume and mass of the calcium content in the cylinders were calculated and compared with the true values. The Agatston score was also evaluated. The Agatston score and mass of calcium obtained at 50 mAs using FBP were 656.92 and 159.91 mg, respectively. In contrast, those obtained at 25 mAs using iDose were 641.91 and 159.05 mg, respectively. No significant differences were found in the calcium measurements obtained using FBP and iDose. In addition, the Agatston score and mass of calcium obtained at 15 mAs and 20 mAs using iDose were not significantly different from those obtained at 25 mAs with iDose. By using iDose, accurate quantification of calcium in the coronary artery can be achieved at 15 mAs using 64-detector CT. The radiation dose can be significantly reduced in coronary artery calcium scoring without impairing the detection and quantification of coronary calcification.

The odontogenic differentiation of human dental pulp stem cells on nanofibrous poly(l-lactic acid) scaffolds in vitro and in vivo

The aim of this study was to investigate the odontogenic differentiation of human dental pulp stem cells (DPSCs) on nanofibrous (NF)-poly(l-lactic acid) (PLLA) scaffolds in vitro and in vivo. Highly porous NF-PLLA scaffolds which mimic the architecture of collagen type I fibers were fabricated by the combination of a phase-separation technique and a porogen-leaching method. The human DPSCs were then seeded onto the scaffolds and cultured in different media for odontogenic differentiation: "Control" medium without supplements; "DXM" medium containing 10(-8)M dexamethasone (DXM), 50 microgml(-1) ascorbic acid and 5mM beta-glycerophosphate; "BMP-7+DXM" medium containing 10(-8)M DXM, 50 microgml(-1) ascorbic acid, 5mM beta-glycerophosphate plus 50 ngml(-1) bone morphogenetic protein 7 (BMP-7). For odontogenic differentiation study in vitro, alkaline phosphatase activity quantification, reverse transcription polymerase chain reaction, scanning electron microscopy, von Kossa staining and calcium content quantification were carried out. While both "DXM" medium and "BMP-7+DXM" medium induced the DPSCs to odontoblast-like cells, the "BMP-7+DXM" medium had greater inducing capacity than the "DXM" medium. Consistent with the in vitro studies, the "BMP-7+DXM" group presented more extracellular matrix and hard tissue formation than the "DXM" group after 8 weeks of ectopic implantation in nude mice. Differentiation of DPSCs into odontoblast-like cells was identified by the positive immunohistochemical staining for dentin sialoprotein. In conclusion, odontogenic differentiation of DPSCs can be achieved on NF-PLLA scaffolds both in vitro and in vivo; the combination of BMP-7 and DXM induced the odontogenic differentiation more effectively than DXM alone. The NF-PLLA scaffold and the combined odontogenic inductive factors provide excellent environment for DPSCs to regenerate dental pulp and dentin.

Chondrogenic and osteogenic differentiations of human bone marrow-derived mesenchymal stem cells on a nanofibrous scaffold with designed pore network

The utilization of 3D scaffolds and stem cells is a promising approach to solve the problem of bone and cartilage tissue shortage and to construct osteochondral (cartilage/bone composite) tissues. In this study, 3D highly porous nanofibrous (NF) poly( l-lactic acid) (PLLA) scaffolds fabricated using a phase separation technique were seeded with multi-potent human bone marrow-derived mesenchymal stem cells (hMSCs) and the constructs were induced along osteogenic and chondrogenic development routes in vitro. Histological analysis and calcium content quantification showed that NF scaffolds supported in vitro bone differentiation. SEM observation showed an altered shape for cells cultured on an NF matrix compared with those on smooth films. Consistent with the morphological change, the gene expression of early chondrogenic commitment marker Sox-9 was enhanced on the NF matrix. NF scaffolds were then used to support long-term in vitro 3D cartilaginous development. It was found that in the presence of TGF-β1, cartilage tissue developed on PLLA NF scaffolds, with the cartilage-specific gene expressed, glycosaminoglycan and type II collagen accumulated, and typical cartilage morphology formed. These findings suggest that NF scaffolds can support both bone and cartilage development and are excellent candidate scaffolds for osteochondral defect repair.

Poly(lactide- co-glycolide)/hydroxyapatite composite scaffolds for bone tissue engineering

Biodegradable polymer/bioceramic composite scaffolds can overcome the limitations of conventional ceramic bone substitutes such as brittleness and difficulty in shaping. However, conventional methods for fabricating polymer/bioceramic composite scaffolds often use organic solvents (e.g., the solvent casting and particulate leaching (SC/PL) method), which might be harmful to cells or tissues. Furthermore, the polymer solutions may coat the ceramics and hinder their exposure to the scaffold surface, which may decrease the likelihood that the seeded osteogenic cells will make contact with the bioactive ceramics. In this study, a novel method for fabricating a polymer/nano-bioceramic composite scaffold with high exposure of the bioceramics to the scaffold surface was developed for efficient bone tissue engineering. Poly( d, l-lactic- co-glycolic acid)/nano-hydroxyapatite (PLGA/HA) composite scaffolds were fabricated by the gas forming and particulate leaching (GF/PL) method without the use of organic solvents. The GF/PL method exposed HA nanoparticles at the scaffold surface significantly more than the conventional SC/PL method does. The GF/PL scaffolds showed interconnected porous structures without a skin layer and exhibited superior enhanced mechanical properties to those of scaffolds fabricated by the SC/PL method. Both types of scaffolds were seeded with rat calvarial osteoblasts and cultured in vitro or were subcutaneously implanted into athymic mice for eight weeks. The GF/PL scaffolds exhibited significantly higher cell growth, alkaline phosphatase activity, and mineralization compared to the SC/PL scaffolds in vitro. Histological analyses and calcium content quantification of the regenerated tissues five and eight weeks after implantation showed that bone formation was more extensive on the GF/PL scaffolds than on the SC/PL scaffolds. Compared to the SC/PL scaffolds, the enhanced bone formation on the GF/PL scaffolds may have resulted from the higher exposure of HA nanoparticles at the scaffold surface, which allowed for direct contact with the transplanted cells and stimulated the cell proliferation and osteogenic differentiation. These results show that the biodegradable polymer/bioceramic composite scaffolds fabricated by the novel GF/PL method enhance bone regeneration compared with those fabricated by the conventional SC/PL method.