Calcium Deposition Research Articles

Genetic Mutations in Cell Junction Proteins Associated with Brain Calcification

AbstractIntracerebral calcium deposition, classified into primary familial brain calcification (PFBC) and secondary brain calcification, occurs within the brain parenchyma and vasculature. PFBC manifests with progressive motor decline, dysarthria, and cognitive impairment, with limited treatment options available. Recent research has suggested a link between dysfunction of the blood–brain barrier (BBB) and PFBC, with certain genetic variants potentially affecting neurovascular unit (NVU) function, thereby contributing to BBB integrity disruption and brain calcification. Cell junctions play an indispensable role in maintaining the function of NVUs. The pathogenic mechanisms of PFBC‐causative genes, such as PDGFRB, PDGFB, MYORG, and JAM2, involve NVU disruption. Cell junctions, such as tight junctions, gap junctions, adherens junctions, desmosomes, hemidesmosomes, and focal adhesions, are vital for cell–cell and cell–extracellular matrix connections, maintaining barrier function, cell adhesion, and facilitating ion and metabolite exchange. Several recent studies have highlighted the role of mutations in genes encoding cell junction proteins in the onset and progression of brain calcification and its related phenotypes. This emerging body of research offers a unique perspective for investigating the underlying mechanisms driving brain calcification. In this review, we conducted an examination of the literature reporting on genetic variants in cell junction proteins associated with brain calcification to delineate potential molecular pathways and investigate genotype–phenotype correlations. This approach not only reinforces the rationale for molecular subtyping of brain calcification but also lays the groundwork for the discovery of novel causative genes involved in pathogenesis. © 2024 International Parkinson and Movement Disorder Society.

Fabrication and evaluation of Zn-EGCG-loaded chitosan scaffolds for bone regeneration: From cellular responses to in vivo performance.

We have synthesized a flavonoid metal complex (FMC) by chelating zinc to epigallocatechin-3-gallate (EGCG), a flavonoid present in green tea and incorporated into chitosan (CS) to form 3D constructs by freeze drying method. Scanning electron microscopy characterized The scaffolds for surface morphology and pore dimensions and depicted the presence of interconnected porous network. The scaffolds exhibited optimal pore size (>50μm), facilitating bone tissue ingrowth and neovascularization. Inclusion of Zn-EGCG into CS matrix improved the mechanical property by increasing compressive strength (0.53±0.045MPa) and reducing enzymatic degradation with controlled swelling. In addition, increased protein adsorption was observed during the initial hour, which is crucial for cell attachment. Furthermore, the FMC inclusion promoted exogenous biomineralization of CS scaffolds as early as 4d in simulated body fluid. Indirect cytotoxicity measurements indicated the scaffolds with Zn-EGCG had no toxic effects on mouse mesenchymal stem cells (mMSCs). Under osteogenic environment, the scaffold promoted calcium deposition of mMSCs by upregulation of ALP activity and increased expression of osteoblast differentiation markers such as Runx2, ColI, OC and OPN. We found that the involvement of miR-15b/smurf-1 signalling pathway behind the osteogenic potential of the scaffold. In vivo assessments using the chick embryo CAM assay showed enhanced angiogenesis and confirmed the scaffold's biocompatibility with no toxicity. Additionally, in a zebrafish scale regeneration model, the scaffold enhanced calcium deposition and osteoblast marker expression, aligning with the in vitro findings. Overall, form the study it is clear that the osteogenic potential of the scaffold is as follows chitosan<EGCG-Chitosan<Zn-EGCG-Chitosan.

Exploring Effects and Mechanism of Ingredients of Herba Epimedii on Osteogenesis and Osteoclastogenesis In Vitro.

Herba Epimedii, a commonly used traditional herb, has been proven effective in ameliorating osteoporosis. However, the active ingredients and potential mechanism need further exploration. To screen active ingredients of Herba Epimedii with the effect of ameliorating osteoporosis and to explore their potential mechanisms. TCMSP and Swiss Target Prediction were applied to collect the ingredients of Herba Epimedii and their targets. UniProt, GeneCards, TTD, DisGeNET, and OMIM were adopted to search osteoporosis-related genes. STRING and DAVID were used to perform enrichment analysis. Effects of screened ingredients were evaluated on MC3T3-E1 cells and RAW264.7 cells, respectively. Eleven ingredients were screened by Network Pharmacology. They exerted a promoting effect on MC3T3-E1 cells (10-9-10-5 M). The ingredients didn't significantly affect ALP activity and osteoblastogenesis-related genes. Baohuoside 1, Sagittatoside B, Chlorogenic acid, Cryptochlorogenic acid, and Neochlorogenic acid significantly increased calcium depositions. The ingredients didn't exhibit a dose-dependent inhibition or promotion on RAW264.7 cells. Baohuoside 1, Sagittatoside B, Neochlorogenic acid, Cryptochlorogenic acid, Icariin, Epimedin A, Chlorogenic acid, Sagittatoside A, and Epimedin C suppressed the level of TRACP. Baohuoside 1, Sagittatoside B, Cryptochlorogenic acid, Neochlorogenic acid, Chlorogenic acid, Sagittatoside A, and Icariin decreased the number of multinucleated osteoclastic cells. Baohuoside 1, Sagittatoside B, and Cryptochlorogenic acid could significantly inhibit MMP-9 expression. Neochlorogenic acid, Sagittatoside B, Chlorogenic acid, and Cryptochlorogenic acid promoted MC3T3-E1 differentiation, among which Neochlorogenic acid showed significant promotion in viability, mineralization, and OPN expression. Baohuoside 1, Sagittatoside B, Cryptochlorogenic acid, Neochlorogenic acid, Chlorogenic acid, and Icariin inhibited RAW264.7 differentiation, among which Baohuoside 1 showed significant inhibition on TRACP, multinucleated osteoclastic cells number and MPP-9 expression. The mechanism might relate to the FoxO signaling pathway, MAPK signaling pathway, and TNF signaling pathway.

Methionine supplementation regulates eggshell quality and uterine transcriptome in late-stage broiler breeders

This study aimed to compare the effects of dietary methionine (Met) and 2-hydroxy-4-(methylthio)-butanoate (HMTBA) on the eggshell quality of broiler breeder hens and elucidate the mechanism of Met in improving eggshell quality from the perspectives of eggshell microstructure and shell gland physiological function. A total of 720 WOD188 broiler breeder hens at 40 wk old were assigned to 3 groups, with 8 replicates per group and 30 birds per replicate. Over 7 weeks, birds were fed a basal diet or the same diet supplemented with 0.15% Met or 0.17% HMTBA. Our findings revealed significant improvements in the Met group for egg shape index, shell thickness, breaking strength, and fracture toughness (P < 0.05), whereas the HMTBA group showed no significant improvements (P > 0.05). Met supplementation increased calcium and phosphorus levels in both serum and shell gland tissue (P < 0.05), and enhanced Ca2+ ATPase activity in shell gland tissue (P < 0.05). Histomorphological changes included enhanced mucosal fold dimensions and increased epithelial height in the shell gland (P < 0.05). Met also improved eggshell ultrastructure, resulting in a thicker effective layer and broader mammillae with fewer type B structures (P < 0.05). The mRNA levels for genes regulating eggshell ultrastructure, such as OC-116, CALB1, and ITM2C, were significantly upregulated in the Met group (P < 0.05). Transcriptome analysis identified 248 differentially upregulated genes in the Met group, primarily linked to the non-canonical Wnt/Ca2+ signaling pathway, crucial for calcium ion transport and cellular proliferation. This research highlights that Met supplementation improves eggshell quality by enhancing calcium transport and cellular proliferation in uterine function, particularly through the modulation of WNT11 and CALB1, influencing calcium deposition and ultrastructural development.

Gold Kiwi-Derived Nanovesicles Mitigate Ultraviolet-Induced Photoaging and Enhance Osteogenic Differentiation in Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BM-MSCs) play a crucial role in bone formation through their ability to differentiate into osteoblasts. Aging, however, detrimentally affects the differentiation and proliferation capacities of BM-MSCs, consequently impairing bone regeneration. Thus, mitigating the aging effects on BM-MSCs is vital for addressing bone-related pathologies. In this study, we demonstrate that extracellular nanovesicles isolated from gold kiwi (GK-NVs) protect human BM-MSCs from ultraviolet (UV)-induced photoaging, thereby alleviating aging-related impairments in cellular functions that are crucial for bone homeostasis. Notably, GK-NVs were efficiently taken up by BM-MSCs without causing cytotoxicity. GK-NVs reduced intracellular reactive oxygen species (ROS) levels upon UV irradiation, restoring impaired proliferation and migration capabilities. Furthermore, GK-NVs corrected the skewed differentiation capacities of UV-irradiated BM-MSCs by enhancing osteoblast differentiation, as evidenced by the increased expression in osteoblast-specific genes and the calcium deposition, and by reducing adipocyte differentiation, as indicated by the decreased lipid droplet formation. These findings position GK-NVs as a promising biomaterial for the treatment of bone-related diseases such as osteoporosis.

Lipocalin-2 promotes CKD vascular calcification by aggravating VSMCs ferroptosis through NCOA4/FTH1-mediated ferritinophagy

Vascular calcification (VC) is a common complication of chronic kidney disease (CKD), for which no effective therapies are available. Hyperphosphatemia, a feature of CKD, is a well-known inducer of VC. High phosphate (HP)-induced ferroptosis plays a crucial role in CKD-related VC (CKD-VC), but the mechanisms remain unclear. Lipocalin-2 (LCN2), an iron-trafficking protein, has been implicated in ferroptosis regulation. In the present study, the role of LCN2 as a potential mediator of CKD-VC was investigated. HP-induced LCN2 expression in the arteries of CKD-VC patients, animal models and vascular smooth muscle cells (VSMCs). LCN2 knockout (LCN2KO) mice and wild-type (WT) mice fed with a high adenine and phosphate (AP) diet were studied to explore CKD-VC. Compared with WT mice, LCN2KO mice showed an amelioration of the CKD-VC induced by the AP diet. The inhibition of LCN2 also alleviated HP-induced calcium deposition and phenotypic transition in VSMCs. Conversely, VSMCs-targeted LCN2 overexpression or recombinant LCN2 treatment exacerbated CKD-VC in vivo and in vitro. Mechanistically, nuclear receptor coactivator 4 (NCOA4)/ferritin heavy chain 1 (FTH1)-mediated ferritinophagy-dependent ferroptosis was involved in LCN2-mediated CKD-VC. Under HP conditions, LCN2 interacted with NCOA4, potentially accelerating the degradation of FTH1 and inducing ferroptosis. The inhibition of LCN2 may rescue the degradation of FTH1 and thus ameliorate ferroptosis, significantly suppressing VSMCs calcification. In summary, our study revealed a novel role for LCN2 induced ferritinophagy-dependent ferroptosis in CKD-VC, and targeting LCN2 might be a promising treatment for CKD-VC.

Heterogeneous long-term and seasonal brine evolution, and Artemia fecal pellet controls on Urmia Lake (NW Iran) salt-crust formation and mineralogy

ABSTRACT Urmia Lake resides as a substantial hypersaline lake characterized by notable fluctuations in water salinity, brine composition, and water level over long-term, annual, and seasonal intervals. Extremely rapid water elevation fall (&amp;gt; 7 m) in the last three decades has caused the formation of a salt crust on the lake floor. A manmade stone causeway divided the lake into two relatively deeper northern parts with minimal water inputs and a shallower southern part with maximal river inflows. Restricted water flow through the narrow water passage of the causeway leads to complex salinity processes, brine evolution, and salt-crust formation in Urmia Lake. This research analyzes the ionic composition of lake-sediment and salt-crust pore water, the mineralogy of salt crusts, and the ionic composition of both surface and deep lake waters during both the wet and dry seasons of 2019. The findings indicate that the northern and southern parts of the lake undergo stratification during wet seasons due to significant freshwater input, whereas they become homogenized during dry seasons through progressive evaporative concentration and water mixing. The spatial and temporal variations in the lake brine type (primary Na-Mg-Cl) and ionic composition contribute to the formation of a halite salt crust (NaCl &amp;gt; 97%) with heterogeneous mineralogy and thickness. In Urmia Lake, the variable thickness and mineralogy of the exposed marginal salt crust suggest rapid salt-crust reorganization by annual and seasonal deposition and dissolution processes. Conversely, the submerged central salt crust, with continuous thickening and constant mineralogy, remains unaffected by seasonal variations in brine type and dissolution processes. It is noteworthy to mention that Artemia (a brine shrimp) controls the mineralogy of the lake salt crust through the deposition of calcium and carbonate ions in the form of biochemical fecal pellets.

Establishment of immortalized rabbit bone marrow mesenchymal stem cells and a preliminary study of their osteogenic differentiation capability.

A stable and standardized source of mesenchymal stem cells is a prerequisite for bone repair tissue engineering research and application. We aimed to establish a stable cell line of bone marrow mesenchymal stem cells from New Zealand rabbits and explore their osteogenic differentiation capacity. Primary rabbit bone marrow mesenchymal stem cells (RBMSCs) were isolated and immortalized via retroviral expression of SV40 Large T antigen (LTA). To assess the osteogenic differentiation capacity of the cells invitro, we studied the alkaline phosphatase (ALP) expression level and calcium deposition in bone morphogenetic protein 9 (BMP9)-induced immortalized cells using ALP staining and quantification, as well as alizarin red staining. Ectopic bone formation by the cells was assessed using micro-computed tomography (μCT) and histological examination. The immortalized cell line we established using SV40 LTA, which we termed iRBMSCs, was non-tumorigenic and maintained long-term proliferative activity. We further discovered that BMP9 (MOI = 30) effectively induced the osteogenic differentiation capacity of iRBMSCs invitro, and there was a synergy with GelMA hydrogel in inducing osteogenic differentiation of the iRBMSCs invivo. We confirmed that iRBMSCs are promising as a stable cell line source for bone defect repair engineering.

Mimicking Scaffolds for Osteogenesis Based on Poly(vinyl alcohol) Hydrogel with Hard Calcium Phosphate and Soft Silk Fibroin Particle for Bone Regeneration: Molecular Organization, Morphology, Properties, and In Vitro Evaluation.

In this study, poly(vinyl alcohol) (PVA)/silk fibroin particle (SFP) hydrogel scaffolds are prepared by mixing compound calcium phosphate (CCP) in different weight ratios (0, 4, 8, and 16%) via the repeated freeze-thawing method. The physicochemical characteristics and biological behavior of the hydrogel are evaluated, and the results show a decreased porous structure of the hydrogel composite with a swelling ability upon CCP addition; however, the mechanical strength and degradation rate increase. Cell attachment and growth analyses demonstrate PVA/SFP/CCP as nontoxic to cells. Furthermore, osteogenesis evaluation shows that the group with 8% CCP in PVA/SFP hydrogel exhibits higher cell adhesion and proliferation than other groups. Additionally, the group exhibits better osteogenic performance than the other groups when alkaline phosphatase activity, total protein content, and calcium deposition are compared. Considered as a mixture, the PVA/SFP/CCP hydrogel is promising for bone tissue engineering applications; particularly, PVA/SFP/8% CCP serves as an optimal choice.

A new approach to reducing slagging based on laser modification of heating surfaces: Field tests and mathematical modeling

The article presents the results of field tests of a new approach to reducing slagging of a boiler furnace. The approach involves laser radiation treatment of metal surfaces to obtain a coating with a high degree of passivation and a special texture (microchannel or anisotropic). Due to the texture and formation of the oxide layer, the resistance of the heating surfaces to adhesion of hydrocarbon fuel combustion products, including slag, is increased. Field tests were conducted over 60 days in an operating boiler burning brown coal, the combustion products of which form solid ferrous and sulfate–calcium deposits. The study used scanning microscopy, X-ray fluorescence spectral analysis and mass analysis of slag deposits. The results showed that steel samples with the Microchannels texture formed by laser radiation are characterized by increased resistance to adhesion of brown coal slag. Using the original software code, a mathematical model has been developed to describe the slag deposition process on heat exchange surfaces modified by laser radiation. A numerical simulation was performed under conditions of dynamic change in the thickness of the slag layer on the surface of the heat exchanger pipe to assess the heat transfer characteristics in the “hot flue gases – slag layer – pipe wall – liquid coolant” system. The results showed that the steel surface modified by laser radiation, compared to the untreated surface, is resistant to slagging at the initial stages of interaction with molten ash from different types of fuel. The use of laser-modified surfaces in boiler furnaces will increase the intervals between procedures for cleaning the furnace from slag by up to 64 %. Also, due to the increase in absorbed heat per unit area by 1.6–2.2 times, the use of laser-modified surfaces increases the efficiency of energy production.

The role of fibroblast growth factor 23 in the assessment of complications in chronic kidney disease

Secondary hyperparathyroidism is an early-stage complication of CKD (Chronic Kidney Disease). It is associated with disorders in bone metabolism, calcium deposition in vessels and organs, significantly increased risk of cardiovascular disease, and death. In recent years, an increasingly important role has been attributed to fibroblast growth factor (FGF 23) in regulating calcium/phosphate homeostasis, hyperparathyroidism, and its relation to vascular calcification, and left ventricular hypertrophy. This study conducted with 110 patients with varying degrees of renal insufficiency shows an increase in FGF23 and PTH when there is a decrease in glomerular filtration, a direct dependency between the levels of phosphate and PTH and FGF23, and a lack of such between calcium and PTH and FGF23. Of particular interest is the fact that patients treated with Calcitriol and Paricalcitol show a reduction in PTH levels, yet maintain high levels of FGF23. PTH serves as a marker for the rate of bone remodeling in patients, where elevated levels indicate renal bone disease. The analysis of FGF 23 is crucial in evaluating cardiovascular risk.

The impact of acromial morphology on the localization and size of calcific tendonitis in the rotator cuff.

This study aimed to investigate the association between acromial morphology and the incidence and extent of calcific tendonitis. Ninety-two patients (33 male, 59 female; mean age: 47 ± 9.7) with calcific tendonitis were included in this retrospective study. Critical shoulder angle (CSA), slope angle, lateral acromial angle (LAA), acromion index (AI), acromial type according to Bigliani, and the morphology of the calcific deposits according to Gartner and Heyer were assessed on x-rays. The localization and volume of the calcific deposits were assessed using magnetic resonance imaging (MRI). Patients were divided into 2 groups: CSA < 33° (group 1) and CSA ≥ 33° (group 2). The median CSA was 33.5° (range=23°-51°), lateral acromial angle (LAA) was 83.6° (range=60°-106°), acromial index (AI) was 0.7 (range=0.4-0.9), and slope angle was 24.1° (range=3°-40°). Lateral acromial angle (P=.000) and AI (P=.000) were statistically different between the 2 groups. Critical shoulder angle was correlated with LAA (P=.000) and AI (P=.000); deposit volumes were correlated with slope angle (P=.001), Bigliani type of the acromion (P=.009), and deposit stage according to Gartner and Heyer (P=.004). There was a correlation between deposit localization and its volume; the size of the deposit increased anteriorly (P=.000). This study has shown that CSA failed to quantify a patient's predisposition to calcific tendonitis. However, the findings demonstrate a relationship between the morphological parameters in the sagittal plane, such as acromial slope and deposit volume, which deserve further research. Level III, Prognostic Study.

A COMPARATIVE STUDY BETWEEN DRY NEEDLING AND KINETIC CHAIN ACTIVATION TECHNIQUE WITH SPECIFIED EXERCISE PROGRAMME IN PAINFUL ARC SYNDROME

Objective: Many researcher have reported that painful arc syndrome arises from multiple lesion i. e. minor tear of supraspinatus tendon, supraspinatus tendinitis, calcified deposit in the supraspinatus tendon, subacromial bursitis, crack fracture of greater tuberosity of humerus and bicipital tenosynovitis. In this study, dry needling and KCAT technique with specific exercise programme has been done to reduce pain and improve functioning and their by prevent shoulder pathologies. The objective of the study is. 1) To evaluate the effectiveness of kinetic chain activation in painful arc syndrome. 2) To evaluate the effectiveness of dry needling therapy in painful arc syndrome. 3) To evaluate the effectiveness of a specific exercise program. Methods: 50 patient diagnosed with painful arc syndrome by the physiotherapy department orthopedic department OPD will be initially assessed for the inclusion or exclusion criteria. prior to the treatment procedure patients are oriented to the study and taken informed consent, patient is divided in to two groups(Group A and Group B) and both groups will be assessed for the pre-test parameter. Outcome measure – shoulder pain and disability scale and goniometry is used for measuring pre-test and post-test. Results: The study was significant in reducing pain level and improving function with the pretest. At the end of 6 w treatment program with, both group A (dry needling) and group B(KCAT) showed improvement in painful arc syndrome. Based on results, the study supports research hypothesis that there was a significant improvement in ROM and significant reduction in SPADI score associated with painful arc syndrome. Patient participated in group B shows more significant changes. Conclusion: The study showed that significant improvement in symptoms of group B as compared to group A from the initial level to week 6. At the end of 6 w training programme both group A and group B Shows improvements in symptoms but the result of study supports that group B (KCAT CONCEPT-finger fanning technique) B is more efficient compare to group A(dry needling).

Novel Quantification Protocol for Cardiovascular Calcification Progression Using Longitudinal MicroPET/MicroCT Images.

Micro positron emission tomography (PET) and micro computed tomography (CT) imaging are powerful, ideal research tools for following the progression of cardiovascular calcification. Due to their non-invasive nature, small research animals can be imaged at multiple time points. The challenge lies in the accurate quantification of cardiovascular calcification. Here, we provide a protocol, using images from the later disease stages as a template, to accurately quantify the progression of cardiovascular calcification in longitudinal studies. The protocol involves 1) the alignment of the chest area in multiple images from the same animal during a longitudinal study as the first step, 2) the definition of a region of interest (ROI) situated within the heart and the aorta at the site of larger calcium deposits that become apparent in later images, and 3) simultaneous segmentation and quantification of calcium deposits across all images acquired during the longitudinal study. This streamlined method enhances the accuracy of image analysis in following the progression of cardiovascular calcification by improving the precision of ROI definition and reducing the variability associated with earlier techniques that analyze individual scans independently.

Abstract 4140181: Mechanisms of SGLT-2 Inhibitor Empagliflozin in Attenuating Intramitochondrial Stress and Restoring Mitochondrial Function in Hyperglycemic Cardiomyocyte

Systemic hyperglycemia causes tissue damage and triggers cardiovascular disease (CVD). Sodium-glucose cotransporter-2 inhibitors (SGLT-2i) are a novel class of glucose-lowering agents that have shown unexpected benefits in clinical trials for the treatment of CVDs. We aim to investigate the underlying mechanisms of how SGLT-2 inhibitors alleviate CVDs associated with elevated glucose stress. iPSC-derived cardiomyocytes (iPSC-CM) were incubated with high glucose concentrations for 72 hours. Mitochondrial function in these cardiomyocytes was assessed by flow cytometry with JC-1 staining and ATP luminescence assay. Intracellular reactive oxygen species (ROS) and intramitochondrial calcium stress were measured using CellROX, MitoSOX, and Rhod-2 AM staining. Patch clamp was employed to determine ion current changes in the cardiomyocytes. Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using the Seahorse XFe96 analyzer. In addition, qPCR, Western blot, and DM mouse heart histological analysis were performed to assess the regulation of associated molecules. The results indicated that exposure to a high glucose environment caused cardiomyocyte injury and impaired mitochondrial biosynthesis. Empagliflozin exhibited a beneficial effect on mitochondrial function by reducing ROS production and calcium deposition. It also mitigated the reduction in respiratory OCR of cardiomyocytes induced by high glucose incubation. Furthermore, molecular analysis revealed that Empagliflozin attenuated the dysregulation of mitochondrial calcium channels and biosynthesis by reducing associated gene expression, including Bcl2 , Mfn1 , Mx2 , Oas1 , Ant3 , Mcu , Micu1 , Vdac1 , Ryr2 , and Cypd-ppid . Histological analysis of DM mouse hearts demonstrated that reduced MFN2 and ZBP1 were target molecules for hyperglycemia-induced reduction of calcium channel currents in cardiomyocytes and could be restored by Empagliflozin treatment. This study concludes that high glucose stress diminishes mitochondrial calcium channel regulators MFN2 and ZBP1 in cardiomyocyte, which reduces calcium channel currents and leads to sensitization of cardiomyocyte to arrhythmogenesis, resulting in VT/VF. It provides experimental evidence for the clinical efficacy of Empagliflozin in ameliorating CVDs and managing diabetes-related CVDs.

Qiang Jin Mixture Promotes Osteogenic Differentiation of MC3T3-E1 Cells via BMP2/Smads Pathway and its Network Pharmacology Study.

The study aimed to explore the potential of QiangJin mixture (QJM), a Chinese herbal compound prescription, in regulating MC3T3-E1 cell differentiation and to analyze the ingredients and therapeutic targets of QJM against osteoporosis based on network pharmacology. MC3T3-E1 cells were incubated with different concentrations of QJM-contained rat serum (5, 10, or 20%). After 14days of cell culture, Alizarin Red staining was performed to assess the mineralization ability of osteoblasts. RT-qPCR was used to measure mRNA levels of osteogenesis-related genes. Western blot was conducted to measure protein levels of factors related to the BMP2/Smads pathway. Functional and pathway enrichment of overlapping targets for QJM and osteoporosis were analyzed using gene ontology and KEGG analyses. As shown by experimental results,QJM-contained serumled to calcium deposition, increased expression levels of osteogenesis-related genes, and activated BMP2/Smad/Runx2 signaling in MC3T3-E1 cells. A total of 125 active compounds and 162 disease-related targets were identified. The core targets were MAPK8, TP53, ESR1, STAT3, MAPK3, IL6, NFKB1, JUN, MAPK1 and AKT1. In conclusion,QJM promotes the osteogenic differentiation of MC3T3-E1 cells by activating the BMP2/Smads signaling. Additionally, QJM is an anti-osteoporotic mixture by regulating diverse therapeutic targets and signaling pathways.

A Clinicopathological Study of Scrotal Calcinosis and Literature Review.

Objective. We aimed to investigate the clinicopathological features and pathogenesis of scrotal calcinosis. Methods. Hematoxylin and eosin (HE)-stained slides were performed on 14 patients with scrotal calcinosis, and the clinicopathological characteristics were analyzed together with clinical data. Meanwhile, 81 patients with extrascrotal calcinosis cutis and 7480 patients with epidermoid cysts without calcification in the skin of various parts of the body were collected and analyzed to explore the etiology and pathogenesis of scrotal calcinosis in conjunction with the literature review. Results. All our patients were adult men with a mean age of 38.2 years, with clinical manifestations of multiple scrotal nodules of varying sizes, microscopically characterized by intradermal calcium deposits, of which 3 lesions had a cystic cavity lined with squamous epithelium. Conclusions. Scrotal calcinosis is a rare benign disease that presents clinically as single or multiple hard nodules in the scrotum, usually asymptomatic, and it is histopathologically characterized by intradermal calcium deposition. There is still controversy as to whether it is idiopathic. We believe that scrotal calcinosis is not idiopathic and originates from epidermoid cysts. Prolonged local compression or injury may be one reason for the development of calcification, and perhaps there are other unknown factors contributing to the development of the disease. Surgical excision is the main treatment for this disease with satisfactory therapeutic effectiveness.

Autonomous cortisol secretion promotes vascular calcification in vivo and in vitro under hyperaldosteronism.

Autonomous cortisol secretion (ACS) is not uncommon in patients with primary aldosteronism (PA). However, the cardiovascular burden of ACS due to its dysregulated cortisol secretion remains poorly understood. Thus, we examined the effects of ACS on vascular calcification in a hyperaldosteronism environment in vitro and in vivo. A total of 339 patients with PA with adrenal incidentaloma and low-dose dexamethasone suppression test data (cutoff level: cortisol > 1.8 μg/dL) from a prospectively maintained database were enrolled; abdominal aortic calcification (AAC) scores were quantitatively estimated. Human aortic smooth muscle cells (HAOSMCs) were used as in vitro model of vascular calcification. In this study, 65 of the 339 patients with PA had ACS; 274 did not. Patients with PA/ACS had a higher AAC score (1171.0 ± 2434.0 vs. 489.5 ± 1085.3, P = 0.012) than patients without ACS. ACS was independently associated with AAC score (β = 0.139, P = 0.004) in multivariate analysis, and post-suppression cortisol level was significantly correlated with the AAC score (P = 0.004). In the HAOSMC model, co-treatment with cortisol synergistically stimulated alkaline phosphatase activity and calcium deposition in a hyperaldosteronism environment. The stimulatory effect of cortisol was abolished by the mineralocorticoid receptor (MR) antagonist eplerenone, but not glucocorticoid receptor antagonist mifepristone, indicating a MR-dependent mechanism. In conclusion, the presence of ACS is associated with heavier vascular calcification in patients with PA. Aldosterone and cortisol synergistically activate HAOSMC calcification via MR signaling, via a process that can be attenuated by eplerenone.

OPTIMIZED SOLID LIPID NANOPARTICLES FOR ENHANCED ORAL BIOAVAILABILITY AND OSTEOGENIC EFFECT OF IPRIFLAVONE: FORMULATION, CHARACTERIZATION, AND IN VITRO EVALUATION

Objective: This study aimed to enhance the oral bioavailability of Ipriflavone (IP) and evaluate its osteogenic effect on human osteosarcoma cells (MG-63) by developing Ipriflavone-loaded Solid Lipid Nanoparticles (IP-SLN). Methods: IP-SLNs were prepared using a modified solvent evaporation method with probe sonication. Formulation optimization employed Central Composite Design (CCD) with independent variables, including lipid amount, surfactant concentration, and sonication time. Characterization was performed using Transmission Electron Microscopy (TEM). In vitro drug release and ex vivo permeation studies were conducted to assess drug release kinetics and bioavailability. Cytotoxicity, Alkaline Phosphatase (ALP) activity, and calcium deposition studies on MG-63 cells evaluated osteogenic effects. Results: TEM images showed round particles with an average diameter of 43.24±3 nm, a zeta potential of-9.53 mV, and a drug entrapment efficiency of 76.53±1.84%. In vitro drug release from IP-SLN was 79.02% compared to 14.21% from IP after 48 h, following the Korsmeyer-Peppas model and first-order kinetics. Ex vivo permeation of IP-SLN was approximately 2-fold higher than IP dispersion. Cytotoxicity studies revealed no toxicity on MG-63 cells. ALP activity and calcium deposition studies indicated that IP-SLN stimulated osteoblast differentiation, increasing alkaline phosphatase activity and mineralization. Pharmacokinetic studies demonstrated that IP-SLN increased the relative bioavailability by 515% compared to ipriflavone. Conclusion: IP-SLN formulations significantly improved the oral bioavailability and osteogenic effects of ipriflavone on MG-63 cells, suggesting potential for novel therapeutic applications in osteoporosis treatment.

Exploring Metabolic Mechanisms in Calcific Tendinopathy and Shoulder Arthrofibrosis: Insights and Therapeutic Implications.

Rotator cuff calcific tendinopathy and arthrofibrosis of the shoulder (adhesive capsulitis) are debilitating musculoskeletal disorders that significantly impact joint function and impair quality of life. Despite its high prevalence and common clinical presentation, the metabolic mechanisms underlying these conditions characterized by pain, and reduced mobility, remain poorly understood. This review aims to elucidate the role of metabolic processes implicated in the pathogenesis of calcific tendinopathy and shoulder arthrofibrosis. We will be focusing on the mechanistic role of how these processes contribute to disease progression and can direct potential therapeutic targets. Calcific tendinopathy is marked by aberrant calcium deposition within tendons, influenced by disrupted calcium and phosphate homeostasis, and altered cellular responses. Key molecular pathways, including bone morphogenetic proteins (BMPs), Wnt signaling, and transforming growth factor-beta (TGF-β), play crucial roles in the pathophysiology of calcification, calcium imbalance, and muscle fibrosis. In contrast, shoulder arthrofibrosis involves excessive collagen deposition and fibrosis within the shoulder joint capsule, driven by metabolic dysregulation and inflammation. The TGF-β signaling pathway and inflammatory cytokines, such as interleukin-6 (IL-6), are central to the fibrotic response. A comparative analysis reveals both shared and distinct metabolic pathways between these conditions, highlighting the interplay between inflammation, cellular metabolism, extracellular matrix remodeling, calcific deposition, and calcium migration to the glenohumeral joints, resulting in adhesive capsulitis, thereby providing insights into their pathophysiology. This review discusses current therapeutic approaches and their limitations, advocating for the development of targeted therapies that address specific metabolic dysregulations. Future therapeutic strategies focus on developing targeted interventions that address the underlying metabolic dysregulation, aiming to improve patient outcomes and advance clinical management. This review offers a comprehensive overview of the metabolic mechanisms involved in calcific tendinopathy and shoulder arthrofibrosis, providing a foundation for future research and therapeutic development.