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Bone mineralization is initiated by matrix vesicles, small extracellular vesicles secreted by osteoblasts, inducing the nucleation and subsequent growth of calcium phosphate crystals inside. Although calcium ions (Ca2+) are abundant throughout the tissue fluid close to the matrix vesicles, the influx of phosphate ions (PO43−) into matrix vesicles is a critical process mediated by several enzymes and transporters such as ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), ankylosis (ANK), and tissue nonspecific alkaline phosphatase (TNSALP). The catalytic activity of ENPP1 in osteoblasts generates inorganic pyrophosphate (PPi) intracellularly and extracellularly, and ANK may allow the intracellular PPi to pass through the plasma membrane to the outside of the osteoblasts. Although the extracellular PPi binds to growing hydroxyapatite crystals to prevent crystal overgrowth, TNSALP on the osteoblasts and matrix vesicles hydrolyzes PPi into PO43− monomers: the prevention of crystal growth is blocked, and PO43− monomers are supplied to matrix vesicles. In addition, PHOSPHO1 is thought to function inside matrix vesicles to catalyze phosphocoline, a constituent of the plasma membrane, consequently increasing PO43− in the vesicles. Accumulation of Ca2+ and PO43− inside the matrix vesicles then initiates crystalline nucleation associated with the inner leaflet of the matrix vesicles. Calcium phosphate crystals elongate radially, penetrate the matrix vesicle’s membrane, and finally grow out of the vesicles to form calcifying nodules, globular assemblies of needle-shaped mineral crystals retaining some of those transporters and enzymes. The subsequent growth of calcifying nodules appears to be regulated by surrounding organic compounds, finally leading to collagen mineralization.

During endochondral bone formation, growth plate chondrocytes are differentially regulated by various factors and hormones. As the cellular phenotype changes, the composition of the extracellular matrix is altered, including the production and composition of matrix vesicles (MV) and their cargo of microRNA. The regulatory functions of these MV microRNA in the growth plate are still largely unknown. To address this question, we undertook a targeted bioinformatics approach. A subset of five MV microRNA was selected for analysis based on their specific enrichment in these extracellular vesicles compared to the parent cells (miR-1-3p, miR-22-3p, miR-30c-5p, miR-122-5p, and miR-133a-3p). Synthetic biotinylated versions of the microRNA were produced using locked nucleic acid (LNA) and were transfected into rat growth plate chondrocytes. The resulting LNA to mRNA complexes were pulled down and sequenced, and the transcriptomic data were used to run pathway analysis pipelines. Bone and musculoskeletal pathways were discovered to be regulated by the specific microRNA, notably those associated with transforming growth factor beta (TGFβ) and Wnt pathways, cell differentiation and proliferation, and regulation of vesicles and calcium transport. These results can help with understanding the maturation of the growth plate and the regulatory role of microRNA in MV.

Since its characterization two decades ago, the phosphatase PHOSPHO1 has been the subject of an increasing focus of research. This work has elucidated PHOSPHO1's central role in the biomineralization of bone and other hard tissues, but has also implicated the enzyme in other biological processes in health and disease. During mineralization PHOSPHO1 liberates inorganic phosphate (Pi) to be incorporated into the mineral phase through hydrolysis of its substrates phosphocholine (PCho) and phosphoethanolamine (PEA). Localization of PHOSPHO1 within matrix vesicles allows accumulation of Pi within a protected environment where mineral crystals may nucleate and subsequently invade the organic collagenous scaffold. Here, we examine the evidence for this process, first discussing the discovery and characterization of PHOSPHO1, before considering experimental evidence for its canonical role in matrix vesicle–mediated biomineralization. We also contemplate roles for PHOSPHO1 in disorders of dysregulated mineralization such as vascular calcification, along with emerging evidence of its activity in other systems including choline synthesis and homeostasis, and energy metabolism. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Mineralizing cells release a special class of extracellular vesicles known as matrix vesicles (MV), crucial for bone mineralization. Following their release, MV anchor to the extracellular matrix (ECM), where their highly specialized enzymatic machinery facilitates the formation of seed mineral within the MV’s lumen, subsequently releasing it onto the ECM. However, how MV propagate mineral onto the collagenous ECM remains unclear. In this study, we address these questions by exploring the “protein corona” paradigm whereby nanoparticles entering a biological milieu become cloaked by a corona of soluble proteins modifying their biological functions. We isolated native MV from the growth plates of chicken embryos. After removing the protein corona from the native MV using high ionic strength buffer, we obtained shaved MV. Reconstituted MVs were produced by incubating shaved MV with the removed protein corona constituents. Our results show that both the removal and reconstitution of protein corona significantly affect the biochemical and physicochemical properties of MV, resulting in 3 well-defined groups. Shaved MV exhibited an increase in tissue nonspecific alkaline phosphatase (TNAP) activity and a decrease in mineral deposition compared to native MV. Reconstituted MV partially recovered these functions, showing a reduction of TNAP activity and mineral deposition compared to native MV. Furthermore, changes in the protein corona affect the MV ability to anchor to the collagenous ECM, which is crucial for initiating the propagation of the mineral phase within this organic matrix. Proteomic analyses revealed changes in the protein profile of the MV resulting from the removal of the protein corona, indicating that shaved proteins were primarily related to external structural and ECM organization and catabolism. These findings underscore the role of the protein corona in modulating the mineralization capabilities of MV. Understanding these interactions could lead to new therapeutic strategies for enhancing bone repair and regeneration. Lay Summary Mineralization-competent cells produce matrix vesicles (MV) that work as nanoreactors in the initiation of bone mineralization. Once released to the extracellular space, MV interact with molecules present in the biological fluids, adsorbing them onto the vesicles’ surface to form a “protein corona” that can modulate the biological functions of MV. Here we show that the removal of the protein corona affects collagen binding and the enzymatic activity of a crucial MV enzyme, while reconstitution of the protein corona reverses those deficits. Our findings provide novel valuable insights for the development of biomimetic materials for bone regeneration medicine applications. Graphical Abstract Graphical Abstract

The invasion of etched dentinal tubules (DTs) by external substances induces dentin hypersensitivity (DH). The deep and compact occlusion of DTs is highly desirable for treating DH but still challenging due to the limited penetrability and mineralization capacities of most current desensitizers. Matrix vesicles (MVs) participate in the regulation of ectopic mineralization. Herein, ectopic MV analogs are prepared by employing natural cell membranes to endow mineral precursors with natural biointerfaces and integrated biofunctions for stimulating dentin remineralization. The analogs quickly access DTs (> 20 µm) in only 5 min and further penetrate deep into the interior of DTs (an extraordinary ∼ 200 µm) in 7 d. Both in vitro and in vivo studies confirm that the DTs are efficiently sealed by the newly formed minerals (> 50 µm) with excellent resistance to wear and acid erosion, which is significantly deeper than most reported values. After repair, the microhardness of the damaged dentin can be recovered to those of healthy dentin. For the first time, cell membrane coating nanotechnology is used as a facile and efficient therapy for in-depth remineralization of DTs in treating DH with thorough and long-term effects, which provides insights into their potential for hard tissue repair.

Matrix vesicles (MVs) are extracellular membrane-bound vesicles of about ~ 50–200 nm in diameter that play a role in the bio-mineralization process of hard tissue formation. The present review is based on the empirical phenomenon of primary mineralization process via matrix vesicle-mediated mechanism with special reference to crystal ghosts as well as the mechanism on the organic–inorganic relationship between matrix vesicles and crystal ghosts, and the transformation that these structures undergo during bio-mineralization.

Biomineralization is a fundamental process key to the development of the skeleton. The phosphatase orphan phosphatase 1 (PHOSPHO1), which likely functions within extracellular matrix vesicles, has emerged as a critical regulator of biomineralization. However, the biochemical pathways that generate intravesicular PHOSPHO1 substrates are currently unknown. We hypothesized that the enzyme ectonucleotide pyrophosphatase/phosphodiesterase 6 (ENPP6) is an upstream source of the PHOSPHO1 substrate. To test this, we characterized skeletal phenotypes of mice homozygous for a targeted deletion of Enpp6 (Enpp6−/−). Micro‐computed tomography of the trabecular compartment revealed transient hypomineralization in Enpp6−/− tibias (p < 0.05) that normalized by 12 weeks of age. Whole‐bone cortical analysis also revealed significantly hypomineralized proximal bone in 4‐ but not 12‐week‐old Enpp6−/− mice (p < 0.05) compared with WT animals. Back‐scattered SEM revealed a failure in 4‐week‐old trabecular bone of mineralization foci to propagate. Static histomorphometry revealed increased osteoid volume (p > 0.01) and osteoid surface (p < 0.05), which recovered by 12 weeks but was not accompanied by changes in osteoblast or osteoclast number. This study is the first to characterize the skeletal phenotype of Enpp6−/− mice, revealing transient hypomineralization in young animals compared with WT controls. These data suggest that ENPP6 is important for bone mineralization and may function upstream of PHOSPHO1 as a novel means of generating its substrates inside matrix vesicles. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

AimsMacrocalcification and microcalcification present different clinical risks, but the regulatory of their formation was unclear. Therefore, this study explored the underlying mechanisms of macrocalcification and microcalcification in diabetes mellitus.MethodsAnterior tibial arteries of amputated diabetic feet were collected. According to the calcium content, patients were divided into less-calcification group and more-calcification group. And calcification morphology in plaques was observed. For further study, an in vivo mouse diabetic atherosclerosis model and an in vitro primary mouse aortic smooth muscle cell model were established. After the receptors for AGEs (RAGE) or galectin-3 were silenced, calcified nodule sizes and sortilin expression were determined. Scanning electron microscopy (SEM) was performed to detect the aggregation of matrix vesicles with the inhibition or promotion of sortilin.ResultsBoth macro- and microcalcification were found in human anterior tibial artery plaques. Macrocalcification formed after the silencing of RAGE, and microcalcification formed after the silencing of galectin-3. In the process of RAGE- or galcetin-3-induced calcification, sortilin played an important role downstream. SEM showed that sortilin promoted the aggregation of MVs in the early stage of calcification and formed larger calcified nodules.ConclusionRAGE downregulated sortilin and then transmitted microcalcification signals, whereas galectin-3 upregulated sortilin, which accelerated the aggregation of MVs in the early stage of calcification and mediated the formation of macrocalcifications, These data illustrate the progression of two calcification types and suggest sortilin as a potential target for early intervention of calcification and as an effective biomarker for the assessment of long-term clinical risk and prognosis.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles carrying various types of molecules. These EV cargoes are often used as pathophysiological biomarkers and delivered to recipient cells whose fates are often altered in local and distant tissues. Classical EVs are exosomes, microvesicles, and apoptotic bodies, while recent studies discovered autophagic EVs, stressed EVs, and matrix vesicles. Here, we classify classical and new EVs and non-EV nanoparticles. We also review EVs-mediated intercellular communication between cancer cells and various types of tumor-associated cells, such as cancer-associated fibroblasts, adipocytes, blood vessels, lymphatic vessels, and immune cells. Of note, cancer EVs play crucial roles in immunosuppression, immune evasion, and immunotherapy resistance. Thus, cancer EVs change hot tumors into cold ones. Moreover, cancer EVs affect nonimmune cells to promote cellular transformation, including epithelial-to-mesenchymal transition (EMT), chemoresistance, tumor matrix production, destruction of biological barriers, angiogenesis, lymphangiogenesis, and metastatic niche formation.

Vascular calcification (VC), which is categorized by intimal and medial calcification, depending on the site(s) involved within the vessel, is closely related to cardiovascular disease. Specifically, medial calcification is prevalent in certain medical situations, including chronic kidney disease and diabetes. The past few decades have seen extensive research into VC, revealing that the mechanism of VC is not merely a consequence of a high-phosphorous and -calcium milieu, but also occurs via delicate and well-organized biologic processes, including an imbalance between osteochondrogenic signaling and anticalcific events. In addition to traditionally established osteogenic signaling, dysfunctional calcium homeostasis is prerequisite in the development of VC. Moreover, loss of defensive mechanisms, by microorganelle dysfunction, including hyper-fragmented mitochondria, mitochondrial oxidative stress, defective autophagy or mitophagy, and endoplasmic reticulum (ER) stress, may all contribute to VC. To facilitate the understanding of vascular calcification, across any number of bioscientific disciplines, we provide this review of a detailed updated molecular mechanism of VC. This encompasses a vascular smooth muscle phenotypic of osteogenic differentiation, and multiple signaling pathways of VC induction, including the roles of inflammation and cellular microorganelle genesis.