Epigenetic Regulation in calcific aortic valve disease: Mechanisms and therapeutic potential.

Beyond tendon interfaces - Functional divergence of the matricellular protein SPARC.

FAM20C and FAM20A in normal and ectopic mineralization: A focus on oro-renal syndromes.

Calcification of dystrophic skeletal muscles was described previously and attributed, among others, to ER-stress, elevated phosphate concentration and chronic inflammation. Tauroursodeoxycholic acid (TUDCA) is considered an artificial chaperone protecting cells against ER-stress thus could prevent an ectopic mineralisation of soft tissues. Because an enhanced ER-stress is a feature of dystrophic muscles and it promotes soft tissue mineralisation we hypothesised that TUDCA treatment should reduce mineral deposits in dystrophic skeletal muscles, and tested this concept using two mouse models of DMD. Four-week old mdx, mdxβetageo and w/t mice were administered TUDCA in drinking water for 4 weeks. At 8 weeks, following tissue-clearing and calcium minerals staining with alizarin, mineralisation was evaluated using whole body scanning. Additionally, isolated skeletal muscles were analysed by Western blotting for ER-stress and calcification markers, and using various microscopic methods. Enzymatic activity of alkaline phosphatase was also assayed. Unexpectedly, TUDCA enhanced calcification of dystrophic but not dystrophin-positive muscles. TUDCA did not affect the elevated ER-stress markers found in dystrophic muscles nor impact pro-calcifying proteins RUNX2, Osterix and BMP2/4, which were also overexpressed in dystrophic muscles. The alkaline phosphatase levels, which were reduced in dystrophic muscles, were not affected by this treatment. The increase in ectopic calcification in dystrophic muscles induced by TUDCA is specific to muscles lacking dystrophin. This effect is not linked to the alleviation of ER stress or the overexpression of proteins directly involved in calcium mineral accumulation.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-21534-0.

This study presents the development of nanocomposite hydrogels that integrate functionalized reduced graphene oxide (rGO) derivatives carboxylated (CBX) and aminated (AMN) in combination with gellan gum, gelatin, and cellulose nanofibrils (CNFs) to enhance osteogenesis for bone tissue engineering. Their synthesis involves ultrasound exfoliation, genipin crosslinking, and freeze‐drying. Micro‐computed tomography (µCT) reveals highly porous, interconnected architectures facilitating nutrient diffusion and cell infiltration. CBX/AMN scaffolds exhibit superior hydration capacity and a biphasic degradation profile. Spectroscopically, enhanced interfacial interactions were confirmed, contributing to the overall structural stability of the hydrogels. In vitro, all scaffolds are cytocompatible, sustain cell viability, and promote osteogenic differentiation consistent with adaptive cellmaterial interactions. Subcutaneous implantation in mice confirms scaffold biocompatibility, showing no signs of inflammation or foreign body response. Histological and immunofluorescence analyses reveal osteogenic potential, with enhanced mineralization and elevated expression of osteopontin and osteocalcin. µCT imaging of explants quantifies ectopic mineralization, spatial deposition patterns, and bone mineral density, correlating scaffold architecture and composition with osteogenic performance. These findings suggest that the synergistic integration of functionalized rGO, CNFs, and biopolymers results in scaffolds with enhanced structural and biological properties, highlighting their potential for design optimization and clinical translation in bone regenerative medicine.

Heterotopic ossification (HO) refers to the pathological formation of bone in soft tissues, typically following trauma, surgical procedures, or as a result of genetic disorders. Notably, injuries to the central nervous system significantly increase the risk of HO, a condition referred to as neurogenic HO (NHO). This review outlines the cellular and molecular mechanisms driving HO, focusing on the inflammatory response, progenitor cell reprogramming, and current treatment strategies. HO is primarily fuelled by a prolonged and dysregulated inflammatory response, characterized by sustained expression of osteoinductive cytokines secreted by M1 macrophages. These cytokines promote the aberrant differentiation of fibro-adipogenic progenitor cells (FAPs) into osteoblasts, leading to ectopic mineralization. Additional factors such as hypoxia, BMP signalling, and mechanotransduction pathways further contribute to extracellular matrix (ECM) remodelling and osteogenic reprogramming of FAPs. In the context of NHO, neuroendocrine mediators enhance ectopic bone formation by influencing both local inflammation and progenitor cell fate decisions. Current treatment options such as nonsteroidal anti-inflammatory drugs (NSAIDs), radiation therapy, and surgical excision offer limited efficacy and are associated with significant risks. Novel therapeutic strategies targeting inflammation, neuropeptide signalling, and calcium metabolism may offer more effective approaches to preventing or mitigating HO progression.

Pseudoxanthoma elasticum is a rare inherited disorder marked by abnormal calcium phosphate deposition in soft connective tissues, particularly the skin, arteries, and eyes. It is caused by inactivating mutations in the ABCC6 gene, which encodes a hepatic efflux transporter. Loss of ABCC6 function leads to reduced plasma levels of pyrophosphate, a key inhibitor of calcification, thereby promoting ectopic mineralization. Oral pyrophosphate therapy has emerged as a potential treatment, but its effectiveness is uncertain. Most ingested pyrophosphate is hydrolyzed in the gut to inorganic phosphate, which may worsen calcification. Moreover, its impact on mineralized tissues remains largely unexplored. Abcc6−/− mice closely mimic human pseudoxanthoma elasticum and are widely used in preclinical studies. Although patients are most concerned about ocular complications, eye calcification is rarely assessed in translational studies using Abcc6−/− mice. Using microcomputed tomography we found that ectopic calcification at the ciliary margin is a reliable marker of ocular disease progression in these mice. Administering pyrophosphate in drinking water at concentrations up to 90 mM did not increase calcification in skin or eyes. However, only very high doses effectively prevented ectopic calcification – doses that would equate to an impractical 2.5 g/kg/day of disodium pyrophosphate in humans. These high doses also led to pyrophosphate accumulation in bone and negatively affected bone structure and strength. In summary, only supraphysiological doses of orally administered pyrophosphate inhibited ectopic calcification in Abcc6−/− mice, but these doses are not feasible for human use and may compromise bone function. These data are especially important considering the currently ongoing clinical trial evaluating the safety and efficacy of oral pyrophosphate administration as a treatment for pseudoxanthoma elasticum.

IntroductionThis study investigates the relationship between mineral content and mechanical properties in collagenous tissues using a mesoscopic model. Unlike previous studies that assumed uniform mineral distributions, our model mimics the impact of combined intrafibrillar and extrafibrillar progressive mineralization on the ligament insertion using a realistic mineral gradient. To our knowledge, this is the first study on a minerally graded region that combines both mineral phases within a mesoscopic Molecular Dynamics framework.MethodsA collagen fibril model is constructed, and Molecular Dynamics (MD) simulations are performed at five equidistant locations along the insertion to analyze the influence of mineralization on collagen fibrils. The model captures the real randomness in mineral cluster size and distribution, improving its accuracy.ResultsResults show that while Young's modulus and ultimate tensile strain remain relatively unchanged, ultimate tensile strength, yield strain, and yield strength are significantly affected by the presence of the mineral content. These changes are mainly caused by the interatomic bonds that restrain the collagen molecular sliding within the fibril.DiscussionClinically, this research sheds light on the mechanical role that the progressive mineral gradient plays in load transfer and stress distribution. It also lays the ground for exploring the effects of aging and other pathological conditions such as ectopic mineralization or calcific tendinopathy, which alter the natural mineral gradient and increase the risk of tissue failure.

Bone morphogenetic proteins (BMPs) are widely recognized for their therapeutic efficacy in bone regeneration, but one side effect of these therapies is ectopic mineralization. Previous work identified a mineral-binding peptide (pVTK, VTKHNLQI(pS)Q(pS)Y; where pS denotes a phosphoserine) with the ability to inhibit mineralization in osteoblasts. This study investigated the application of pVTK for inhibiting ectopic mineralization secondary to BMP delivery invitro and invivo. It was hypothesized that a mineral binding peptide could be delivered alongside BMP to limit unwanted mineralization without limiting the pro-osteogenic effects of the BMP signaling pathway. Invitro, pVTK reduced BMP-stimulated mineral deposition in an osteoblast cell line, as determined by a significant reduction in extracellular matrix calcium deposition with > 300 μM pVTK (p < 0.0001) (at 50 ng/mL BMP2). Importantly, pVTK inhibited mineral deposition without competing with the BMP ligand or diminishing the osteogenic phenotype of the cells in response to BMP stimulation, as demonstrated by no changes in intracellular/extracellular osteogenic protein levels with addition of pVTK. Invivo, pVTK reduced ectopic mineralization of BMP-loaded subcutaneous implants by 92% (p = 0.0101) compared to PBS-treated controls. In an acellular model of spontaneous mineralization, pVTK disrupted mineral deposition and reduced crystallinity and crystal organization (as measured via Raman spectroscopy), demonstrating that pVTK is not solely reliant on cell mechanisms for inhibiting mineralization. These findings support the use of a mineral binding peptide for controlling ectopic mineralization secondary to BMP therapies without interfering with the BMP osteogenic pathway, which is necessary for a regenerative effect.

The craniofacial, dental and systemic manifestations of Enamel Renal Syndrome: A Scoping review.

Pseudoxanthoma elasticum (PXE) is an inherited mineralization disorder that leads to calcification of elastic fibers. The ocular characteristics arise due to the calcification of Bruch's membrane, which is located between the retinal pigment epithelium and the choriocapillaris. Despite major scientific progress in understanding the underlying pathological mechanism in recent years, no causal treatment has so far been established. Therefore, the management of patients is currently limited to the treatment of secondary complications, such as intravitreal anti-vascular endothelial growth factor (VEGF) injections in cases of exudative neovascularization. This article discusses upcoming studies that aim to reduce ectopic mineralization. To confirm the diagnosis and possible inclusion in such studies asecond assessment should therefore be carried out in aspecialized center for PXE.

Relevance In the human body, pathological conditions are often accompanied by ectopic mineralization. The mineral composition of the trabecular meshwork is not reliably known, partly due to the technical difficulty of obtaining and preserving tissue sections [1, 2]. This study is aimed at determining the presence of a bioinorganic component in the drainage zone of the eye in glaucoma [3]. Objective Evaluation of mineralization processes occurring in the anterior chamber of the eye in open-angle glaucoma. Material and methods Using an EVO LS 10 scanning electron microscope (Zeiss, Germany) with an Oxford X-Max-50 energy-dispersive X-ray spectrometer (Oxford, UK), an analysis of the chemical composition of trabecular tissue preparations from 29 patients (30 eyes) aged 73 (58; 78) years with stage II–III open-angle glaucoma, as well as the pigment sheet of the iris obtained from 5 cadaver eyes, was performed. The surface of all samples was visualized in SEM in low vacuum mode (EP, 70 Pa) using a backscattered electron detector (BSE) at an accelerating voltage of 21.5 kV and a sample current of 20–60 pA. Gross chemical microanalysis and micromapping were performed for selected chemical elements: C, N, O, Ca, Cl P, Si, S. Results Significant mineral obliteration of the drainage zone tissue was detected in 7 out of 30 samples: silitization in 3 samples, calcification in 4 samples. The effect of autonomous calcification of pigment granules was revealed: when simulating vital conditions, a mineral phase stoichiometrically corresponding to dicalcium phosphate dihydrate (DCPD) and amorphous calcium phosphate (ACP) was detected on the surface of the granules [4, 5]. Conclusion Calcium and silica obliteration is a potentially significant factor in fluid retention at the trabecular level. Acid-base imbalance in the trabecular region has been identified, contributing to the occurrence of mineral obliteration. Keywords: open-angle glaucoma; biomineralization; scanning electron microscopy

Chronic kidney disease (CKD) impacts a large percentage of the global population. Chronic kidney disease-mineral bone disorder (MBD) is the broad term describing alterations in key circulating factors involved in mineralization, ectopic calcification, and bone abnormalities. Cardiovascular complications, involving vascular calcification are one of the leading causes of death in this patient population. Plasma levels of pyrophosphate, a potent inhibitor of ectopic mineralization, are low in CKD and end-stage kidney disease patients. These data suggest that the correction of pyrophosphate levels could stand out as a crucial therapeutic goal to mitigate vascular calcifications and reduce cardiovascular mortality. The primary enzyme responsible for the generation of plasma pyrophosphate is ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We therefore evaluated INZ-701, a recombinant human ENPP1-Fc fusion protein, in an adenine-induced rat model of CKD. Our investigation revealed that INZ-701 administration resulted in significantly lower levels of calcification in the vasculature and soft tissues. Moreover, INZ-701 treatment significantly prevented the osteoid volume increase observed in vehicle-treated rats, addressing another critical clinical manifestation of CKD-MBD. These results underscore the potential of INZ-701 to reduce vascular calcification and bone mineralization abnormalities in CKD.

Collagen-mediated cardiovascular calcification.

Thalassemia, once associated with limited survival, now sees extended life expectancy due to treatment advancements, but new complications such as pseudoxanthoma elasticum (PXE)-like syndrome are emerging. In fact, thalassemia patients develop PXE-like features more frequently than the general population. These features include skin lesions, ocular changes, and vascular issues like arterial calcifications, all linked to oxidative damage from iron overload. PXE-like syndrome in thalassemia mimics inherited PXE but is acquired. The underlying cause is thought to be oxidative stress due to iron overload, which induces free radicals and damages elastic tissues. Unlike inherited PXE, this form does not involve mutations in the ABCC6 gene, suggesting different pathogenic mechanisms, including abnormal fibroblast metabolism and oxidative processes. The vascular calcification seen in this syndrome often follows elastic fiber degeneration, with proteoglycans and glycoproteins acting as nucleation sites for mineralization. The condition can lead to severe cardiovascular and gastrointestinal complications. Studies have shown a significant incidence of PXE-like skin lesions in thalassemia patients, with some dying from cardiovascular complications. Research on ABCC6, a transporter protein involved in ectopic mineralization, has highlighted its role in various conditions, including PXE, beta-thalassemia, and generalized arterial calcification of infancy. ABCC6 mutations or reduced expression led to ectopic mineralization, affecting cardiovascular, ocular, and dermal tissues. The exact molecular mechanisms linking ABCC6 deficiency to ectopic mineralization remain unclear, though it is known to influence calcification-modulating proteins. This review focuses on the role of ABCC6 in the pathogenesis of calcifications, especially intracranial vascular calcifications in PXE and beta-thalassemia.

Brain calcification, the ectopic mineral deposits of calcium phosphate, is a frequent radiological finding and a diagnostic criterion for primary familial brain calcification. We previously showed that microglia curtail the growth of small vessel calcification via the triggering receptor expressed in myeloid 2 (TREM2) in the Pdgfb ret/ret mouse model of primary familial brain calcification. Because boosting TREM2 function using activating antibodies has been shown to be beneficial in other disease conditions by aiding in microglial clearance of diverse pathologies, we investigated whether administration of a TREM2-activating antibody could mitigate vascular calcification in Pdgfb ret/ret mice. Single-nucleus RNA-sequencing analysis showed that calcification-associated microglia share transcriptional similarities to disease-associated microglia and exhibited activated TREM2 and TGFβ signaling. Administration of a TREM2-activating antibody increased TREM2-dependent microglial deposition of cathepsin K, a collagen-degrading protease, onto calcifications. However, this did not ameliorate the calcification load or alter the mineral composition and the microglial phenotype around calcification. We therefore conclude that targeting microglia with TREM2 agonistic antibodies is insufficient to demineralize and clear vascular calcifications.

Calciprotein particles (CPPs) are an endogenous buffering system, clearing excessive amounts of Ca2+ and PO43- from the circulation and thereby preventing ectopic mineralization. CPPs circulate as primary CPPs (CPP1), which are small spherical colloidal particles, and can aggregate to form large, crystalline, secondary CPPs (CPP2). Even though it has been reported that CPPs are toxic to vascular smooth muscle cells (VSMC) in vitro, their effect(s) on the vasculature remain unclear. Here we have shown that CPP1, but not CPP2, increased arterial stiffness ex vivo. Interestingly, the effects were more pronounced in the abdominal infrarenal aorta compared to the thoracic descending aorta. Further, we demonstrated that CPP1 affected both endothelial and VSMC function, impairing vasorelaxation and contraction respectively. Concomitantly, arterial glycosaminoglycan accumulation was observed as well, which is indicative of an increased extracellular matrix stiffness. However, these effects were not observed in vivo. Hence, we concluded that CPP1 can induce vascular dysfunction.

Ectopic mineralization, or pathologic calcifications, occur outside the skeletal system. While rare in adults, nasal foreign bodies are common in children and mentally disabled patients. Often asymptomatic, they can remain unnoticed for years. This article presents an unusual case of a nasal foreign body in a 46-year-old male, incidentally discovered during routine dental radiography. The patient complained of tooth pain and sensitivity, with a history of sinusitis and hypertension. Radiographic examination revealed a radiopaque lesion in the right nasal cavity, causing deviation of the nasal septum. A clinical diagnosis of a foreign body was made, considering differential diagnoses such as rhinolith or parasitic infection. This case underscores the importance of thorough examination and attention to detail by dentists. Improved communication and careful radiograph interpretation can lead to the discovery of unexpected findings, facilitating accurate diagnosis and treatment.

Generalized Arterial Calcification of Infancy (GACI)

Dental pulp stem cells (DPSCs) are responsible for maintaining pulp structure and function after pulp injury. DPSCs migrate directionally to the injury site before differentiating into odontoblast-like cells, which is a prerequisite and a determinant in pulp repair. Increasing evidence suggests that sensory neuron-stem cell crosstalk is critical for maintaining normal physiological functions, and sensory nerves influence stem cells mainly by neuropeptides. However, the role of sensory nerves on DPSC behaviors after pulp injury is largely unexplored. Here, we find that sensory nerves released significant amounts of calcitonin gene-related peptide (CGRP) near the injury site, acting directly on DPSCs via receptor activity modifying protein 1 (RAMP1) to promote collective migration of DPSCs to the injury site, and ultimately promoting pulp repair. Specifically, sensory denervation leads to poor pulp repair and ectopic mineralization, in parallel with that DPSCs failed to be recruited to the injury site. Furthermore, in vitro evidence shows that sensory nerve-deficient microenvironment suppressed DPSC migration prominently among all related behaviors. Mechanistically, the CGRP-Ramp1 axis between sensory neurons and DPSCs was screened by single-cell RNA-seq analysis and immunohistochemical studies confirmed that the expression of CGRP rather than Ramp1 increases substantially near the damaged site. We further demonstrated that CGRP released by sensory nerves binds the receptor Ramp1 on DPSCs to facilitate cell collective migration by an indirect co-culture system using conditioned medium from trigeminal neurons, CGRP recombinant protein and antagonists BIBN4096. The treatment with exogenous CGRP promoted the recruitment of DPSCs, and ultimately enhanced the quality of pulp repair. Targeting the sensory nerve could therefore provide a new strategy for stem cell-based pulp repair and regeneration.