The Na+/Ca2+ exchanger NCX3 mediates Ca2+ entry into matrix vesicles to facilitate initial steps of mineralization in osteoblasts.

Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na+/Ca2+ exchanger isoform-3 (NCX3, SLC8A3) was presumed to function as major Ca2+ transporter responsible for Ca2+ extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV-mediated mineralization process and its impact on bone formation. Using differentiated MC3T3-E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca2+ deposition due to reduced Ca2+ entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3-/-) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, µCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3-/- mice, thus supporting the critical role of NCX3 in facilitating Ca2+ uptake into the MVs to initiate osteoblast-mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization-related disorders.

Preparation of black phosphorus@sodium alginate microspheres with bone matrix vesicle structure via electrospraying for bone regeneration.

Bone matrix vesicles are commonly acknowledged as the primary site of biomineralization in human skeletal tissue. Black phosphorus has exhibited favorable properties across various chemical and physical domains. In this investigation, a novel composite microsphere was synthesized through the amalgamation of sodium alginate (ALG) with black phosphorus nanosheets (BP) utilizing the electrospray (ES) technique. These microspheres were tailored to mimic the regulatory function of matrix vesicles (MV) upon exposure to a biomimetic mineralization fluid (SBF) during the biomineralization process. Results revealed that black phosphorus nanosheets facilitated the generation of hydroxyapatite (HA) on the microsphere surface. Live-dead assays and cell proliferation experiments showcased a cell survival rate exceeding 85 %. Moreover, wound healing assessments unveiled that M-ALG-BP microspheres exhibited superior migration capacity, with a migration rate surpassing 50 %. Furthermore, after 7 days of osteogenic induction, M-ALG-BP microspheres notably stimulated osteoblast differentiation. Particularly noteworthy, M-ALG-BP microspheres significantly enhanced osteogenic differentiation of osteoblasts and induced collagen production in vitro. Additionally, experiments involving microsphere implantation into mouse skeletal muscle demonstrated the potential for ectopic mineralization by ALG-BP microspheres. This investigation underscores the outstanding mineralization properties of ALG-BP microspheres and their promising clinical prospects in bone tissue engineering.

Additional Insights Into the Role of Osteocalcin in Osteoblast Differentiation and in the Early Steps of Developing Alveolar Process of Rat Molars.

This study investigated whether osteocalcin (OCN) is present in osteoblast precursors and its relationship with initial phases of alveolar process formation. Samples of maxillae of 16-, 18-, and 20-day-old rat embryos (E16, E18, and E20, respectively), and 05-, 10-, and 15-day-old postnatal rats (P05, P10, and P15, respectively) were fixed and embedded in paraffin or araldite. Immunohistochemistry for osterix (Osx), alkaline phosphatase (ALP), and OCN detection was performed and the number of immunolabelled cells was computed. Non-decalcified sections were subjected to the von Kossa method combined with immunohistochemistry for Osx or OCN detection. For OCN immunolocalization, samples were fixed in 0.5% glutaraldehyde/2% formaldehyde and embedded in LR White resin. The highest number of ALP- and OCN-immunolabelled cells was observed in dental follicle of E16 specimens, mainly in basal portions of dental alveolus. In corresponding regions, osteoblasts in differentiation adjacent to von Kossa-positive bone matrix exhibited Osx and OCN immunoreactivity. Ultrastructural analysis revealed OCN immunoreactive particles inside osteoblast in differentiation, and in bone matrix associated with collagen fibrils and within matrix vesicles, at early stages of alveolar process formation. Our results indicate that OCN plays a role in osteoblast differentiation and may regulate calcium/phosphate precipitation during early mineralization of the alveolar process.

Altered Caveolin-1 Dynamics Result in Divergent Mineralization Responses in Bone and Vascular Calcification.

Introduction: Though vascular smooth muscle cells adopt an osteogenic phenotype during pathological vascular calcification, clinical studies note an inverse correlation between bone mineral density and arterial mineral-also known as the calcification paradox. Both processes are mediated by extracellular vesicles (EVs) that sequester calcium and phosphate. Calcifying EV formation in the vasculature requires caveolin-1 (CAV1), a membrane scaffolding protein that resides in membrane invaginations (caveolae). Of note, caveolin-1-deficient mice, however, have increased bone mineral density. We hypothesized that caveolin-1 may play divergent roles in calcifying EV formation from vascular smooth muscle cells (VSMCs) and osteoblasts (HOBs). Methods: Primary human coronary artery VSMCs and osteoblasts were cultured for up to 28 days in an osteogenic media. CAV1 expression was knocked down using siRNA. Methyl ß-cyclodextrin (MßCD) and a calpain inhibitor were used, respectively, to disrupt and stabilize the caveolar domains in VSMCs and HOBs. Results: CAV1 genetic variation demonstrates significant inverse relationships between bone-mineral density (BMD) and coronary artery calcification (CAC) across two independent epidemiological cohorts. Culture in osteogenic (OS) media increased calcification in HOBs and VSMCs. siRNA knockdown of CAV1 abrogated VSMC calcification with no effect on osteoblast mineralization. MßCD-mediated caveolae disruption led to a 3-fold increase of calcification in VSMCs treated with osteogenic media (p < 0.05) but hindered osteoblast mineralization (p < 0.01). Conversely, stabilizing caveolae by calpain inhibition prevented VSMC calcification (p < 0.05) without affecting osteoblast mineralization. There was no significant difference in CAV1 content between lipid domains from HOBs cultured in OS and control media. Conclusion: Our data indicate fundamental cellular-level differences in physiological and pathophysiological mineralization mediated by CAV1 dynamics. This is the first study to suggest that divergent mechanisms in calcifying EV formation may play a role in the calcification paradox. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00779-7.

AnnexinA6: a potential therapeutic target gene for extracellular matrix mineralization.

The mineralization of the extracellular matrix (ECM) is an essential and crucial process for physiological bone formation and pathological calcification. The abnormal function of ECM mineralization contributes to the worldwide risk of developing mineralization-related diseases; for instance, vascular calcification is attributed to the hyperfunction of ECM mineralization, while osteoporosis is due to hypofunction. AnnexinA6 (AnxA6), a Ca2+-dependent phospholipid-binding protein, has been extensively reported as an essential target in mineralization-related diseases such as osteoporosis, osteoarthritis, atherosclerosis, osteosarcoma, and calcific aortic valve disease. To date, AnxA6, as the largest member of the Annexin family, has attracted much attention due to its significant contribution to matrix vesicles (MVs) production and release, MVs-ECM interaction, cytoplasmic Ca2+ influx, and maturation of hydroxyapatite, making it an essential target in ECM mineralization. In this review, we outlined the recent advancements in the role of AnxA6 in mineralization-related diseases and the potential mechanisms of AnxA6 under normal and mineralization-related pathological conditions. AnxA6 could promote ECM mineralization for bone regeneration in the manner described previously. Therefore, AnxA6 may be a potential osteogenic target for ECM mineralization.

Osteoblast-Derived Matrix Vesicles Exhibit Exosomal Traits and a Unique Subset of microRNA: Their Caveolae-Dependent Endocytosis Results in Reduced Osteogenic Differentiation.

Matrix vesicles (MVs) are nano-sized extracellular vesicles that are anchored in the extracellular matrix (ECM). In addition to playing a role in biomineralization, osteoblast-derived MVs were recently suggested to have regulatory duties. The aims of this study were to establish the characteristics of osteoblast-derived MVs in the context of extracellular vesicles like exosomes, assess their role in modulating osteoblast differentiation, and examine their mechanism of uptake. MVs were isolated from the ECM of MG63 human osteoblast-like cell cultures and characterized via enzyme activity, transmission electron microscopy, nanoparticle tracking analysis, Western blot, and small RNA sequencing. Osteoblasts were treated with MVs from two different culture conditions (growth media [GM]; osteogenic media [OM]) to evaluate their effects on the differentiation and production of inflammatory markers and on macrophage polarization. MV endocytosis was assessed using a lipophilic, fluorescent dye and confocal microscopy with the role of caveolae determined using methyl-ß-cyclodextrin. MVs exhibited a four-fold enrichment in alkaline phosphatase specific activity compared to plasma membranes; were 50-150 nm in diameter; possessed exosomal markers CD63, CD81, and CD9 and endosomal markers ALIX, TSG101, and HSP70; and were selectively enriched in microRNA linked to an anti-osteogenic effect and to M2 macrophage polarization. Treatment with GM or OM MVs decreased osteoblast differentiation. Osteoblasts endocytosed MVs using a mechanism that involves caveolae. These results support the hypothesis that osteoblasts produce MVs that participate in the regulation of osteogenesis.

The mechanism of biomineralization: Progress in mineralization from intracellular generation to extracellular deposition.

Biomineralization is a highly regulated process that results in the deposition of minerals in a precise manner, ultimately producing skeletal and dental hard tissues. Recent studies have highlighted the crucial role played by intracellular processes in initiating biomineralization. These processes involve various organelles, such as the endoplasmic reticulum(ER), mitochondria, and lysosomes, in the formation, accumulation, maturation, and secretion of calcium phosphate (CaP) particles. Particularly, the recent in-depth study of the dynamic process of the formation of amorphous calcium phosphate(ACP) precursors among organelles has made great progress in the development of the integrity of the biomineralization chain. However, the precise mechanisms underlying these intracellular processes remain unclear, and they cannot be fully integrated with the extracellular mineralization mechanism and the physicochemical structure development of the mineralization particles. In this review, we aim to focus on the recent progress made in understanding intracellular mineralization organelles' processes and their relationship with the physicochemical structure development of CaP and extracellular deposition of CaP particles.

1α,25-Dihydroxyvitamin D3 Regulates microRNA Packaging in Extracellular Matrix Vesicles and Their Release in the Matrix.

Growth plate chondrocytes are regulated by numerous factors and hormones as they mature during endochondral bone formation, including transforming growth factor beta-1 (TGFb1), bone morphogenetic protein 2 (BMP2), insulin-like growth factor-1 (IFG1), parathyroid hormone and parathyroid hormone related peptide (PTH, PTHrP), and Indian hedgehog (IHH). Chondrocytes in the growth plate's growth zone (GC) produce and export matrix vesicles (MVs) under the regulation of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. 1α,25(OH)2D3 regulates MV enzyme composition genomically and 1α,25(OH)2D3 secreted by the cells acts on the MV membrane nongenomically, destabilizing it and releasing MV enzymes. This study examined the regulatory role 1α,25(OH)2D3 has over production and packaging of microRNA (miRNA) into MVs by GC cells and the release of miRNA by direct action on MVs. Costochondral cartilage GC cells were treated with 1α,25(OH)2D3 and the miRNA in the cells and MVs sequenced. We also treated MVs with 1α,25(OH)2D3 and determined if the miRNA was released. To assess whether MVs can act directly with chondrocytes and if this is regulated by 1α,25(OH)2D3, we stained MVs with a membrane dye and treated GC cells with them. 1α,25(OH)2D3 regulated production and packaging of a unique population of miRNA into MVs compared to the vehicle control population. 1α,25(OH)2D3 treatment of MVs did not release miRNA. Stained MVs were endocytosed by GC cells and this was increased with 1α,25(OH)2D3 treatment. This study adds new regulatory roles for 1α,25(OH)2D3 with respect to packaging and transport of MV miRNAs.

Western Blot Protocols for Analysis of CCN Proteins and Fragments in Exosomes, Vesicle-Free Fractions, and Cells.

Cellular Communication Network (CCN) proteins are growth factors that play key roles in many pathophysiological events, including bone formation, wound healing, and cancer. CCN factors and fragments generated by metalloproteinases-dependent cleavage are often associated with extracellular matrix (ECM) or small extracellular vesicles (sEVs) such as exosomes or matrix-coated vesicles. We provide reliable methods and protocols for Western blotting to analyze CCN factors and fragments in cells, sEVs, and vesicle-free fractions.

Do Media Extracellular Vesicles and Extracellular Vesicles Bound to the Extracellular Matrix Represent Distinct Types of Vesicles?

Mineralization-competent cells, including hypertrophic chondrocytes, mature osteoblasts, and osteogenic-differentiated smooth muscle cells secrete media extracellular vesicles (media vesicles) and extracellular vesicles bound to the extracellular matrix (matrix vesicles). Media vesicles are purified directly from the extracellular medium. On the other hand, matrix vesicles are purified after discarding the extracellular medium and subjecting the cells embedded in the extracellular matrix or bone or cartilage tissues to an enzymatic treatment. Several pieces of experimental evidence indicated that matrix vesicles and media vesicles isolated from the same types of mineralizing cells have distinct lipid and protein composition as well as functions. These findings support the view that matrix vesicles and media vesicles released by mineralizing cells have different functions in mineralized tissues due to their location, which is anchored to the extracellular matrix versus free-floating.

Correction of Vascular Calcification and Hyperphosphatemia in CKD Rats Treated with ASARM Peptide.

Background: Abnormalities in calcium, phosphorus, PTH, vitamin D metabolism, bone, and vascular calcification occur in chronic kidney disease mineral bone disorder (CKD-MBD). Calciphylaxis, involving painful, ulcerative skin lesions, is also a major problem associated with CKD-MBD. There are no quality medical interventions to address these clinical issues. Bone ASARM peptides are strong inhibitors of mineralization and induce hypophosphatemia by inhibiting phosphate uptake from the gut. We hypothesize treatment of CKD-MBD rats with ASARM peptides will reverse hyperphosphatemia, reduce soft-tissue calcification, and prevent calciphylaxis. Methods: To test our hypothesis, we assessed the effects of synthetic ASARM peptide in rats that had undergone a subtotal 5/6th nephrectomy (56NEPHREX), a rodent model of CKD-MBD. All rats were fed a high phosphate diet (2% Pi) to worsen mineral metabolism defects. Changes in serum potassium, phosphate, BUN, creatinine, PTH, FGF23, and calcium were assessed in response to 28 days of ASARM peptide infusion. Also, changes in bone quality, soft-tissue calcification, and expression of gut Npt2b (Slc34a2) were studied following ASARM peptide treatment. Results: Rats that had undergone 56NEPHREX treated with ASARM peptide showed major improvements in hyperphosphatemia, blood urea nitrogen (BUN), and bone quality compared with vehicle controls. Also, ASARM-infused 56NEPHREX rats displayed improved renal, brain, and cardiovascular calcification. Notably, ASARM peptide infusion prevented the genesis of subdermal medial blood vessel calcification and calciphylaxis-like lesions in 56NEPHREX rats compared with vehicle controls. Conclusions: ASARM peptide infusion corrects hyperphosphatemia and improves vascular calcification, renal calcification, brain calcification, bone quality, renal function, and skin mineralization abnormalities in 56NEPHREX rats. These findings confirm our hypothesis and support the utility of ASARM peptide treatment in patients with CKD-MBD.

Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization.

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.

An in silico model for woven bone adaptation to heavy loading conditions in murine tibia.

Existing in silico models for lamellar bone adaptation to mechanical loading are unsuitable for predicting woven bone growth. This anomaly is due to the difference in mechanobiology of the woven bone with respect to that of the lamellar bone. The present study is aimed at developing an in silico bone-adaptation model for woven bone at cellular and tissue levels. The diffusion of Ca2+ ions reaching lining cells from the osteocytic network and the bone cortex in response to a mechanical loading on the cortical bone has been considered as a stimulus. The diffusion of ions within osteocytic network has been computed with a lacunar-canalicular network (LCN) in which bone cells are uniformly arranged. Strain energy density is assumed to regulate ion flow within the network when the induced normal strain is above a threshold level. If the induced strain exceeds another higher threshold level, then the strain with a power constant is additionally assumed to regulate the stimulus. The intracellular flow of Ca2+ ions within the LCN has been simulated using Fick's laws of diffusion, using a finite element method. The ion diffusion from bone cortex to vesicles has been formulated as a normal strain with a power constant. The stimuli reaching the surface cells are assumed to form the new bone. The mathematical model closely predicts woven bone growth in mouse and rat tibia for various in vivo loading conditions. This model is the first to predict woven bone growth at tissue and cellular levels in response to heavy mechanical loading.

The Role of Matrix-Bound Extracellular Vesicles in the Regulation of Endochondral Bone Formation.

Matrix vesicles are key players in the development of the growth plate during endochondral bone formation. They are involved in the turnover of the extracellular matrix and its mineralization, as well as being a vehicle for chondrocyte communication and regulation. These extracellular organelles are released by the cells and are anchored to the matrix via integrin binding to collagen. The exact function and makeup of the vesicles are dependent on the zone of the growth plate in which they are produced. Early studies defined their role as sites of initial calcium phosphate deposition based on the presence of crystals on the inner leaflet of the membrane and subsequent identification of enzymes, ion transporters, and phospholipid complexes involved in mineral formation. More recent studies have shown that they contain small RNAs, including microRNAs, that are distinct from the parent cell, raising the hypothesis that they are a distinct subset of exosomes. Matrix vesicles are produced under complex regulatory pathways, which include the action of steroid hormones. Once in the matrix, their maturation is mediated by the action of secreted hormones. How they convey information to cells, either through autocrine or paracrine actions, is now being elucidated.

Bone-like hydroxyapatite anchored on alginate microspheres for bone regeneration.

Inspired by the initial mineralization process in bone matrix vesicles (MVs), we used Dulbecco's Modified Eagle's Medium (DMEM) to establish the similar physiological environment to that in MVs for biomimetic mineralization on alginate (ALG) microspheres. The results showed that HA crystals were firstly formed and anchored on the membrane of microspheres like the initial deposition of hydroxyapatite crystals inside MVs. With the continuous growth and accumulation of mineral crystals, HA coating was finally formed on ALG microspheres. The mineralized ALG microspheres (M-ALG microspheres) show good biocompatibility and osteogenic performance. The HA coating is also conducive to the active migration of osteoblasts to the surface of M-ALG microspheres. Collectively, bone-like HA crystals anchored on ALG microspheres may provide a good prospect to promote the repair of bone defects.

Matrix Vesicles as a Therapeutic Target for Vascular Calcification.

Vascular calcification (VC) is linked to an increased risk of heart disease, stroke, and atherosclerotic plaque rupture. It is a cell-active process regulated by vascular cells rather than pure passive calcium (Ca) deposition. In recent years, extracellular vesicles (EVs) have attracted extensive attention because of their essential role in the process of VC. Matrix vesicles (MVs), one type of EVs, are especially critical in extracellular matrix mineralization and the early stages of the development of VC. Vascular smooth muscle cells (VSMCs) have the potential to undergo phenotypic transformation and to serve as a nucleation site for hydroxyapatite crystals upon extracellular stimulation. However, it is not clear what underlying mechanism that MVs drive the VSMCs phenotype switching and to result in calcification. This article aims to review the detailed role of MVs in the progression of VC and compare the difference with other major drivers of calcification, including aging, uremia, mechanical stress, oxidative stress, and inflammation. We will also bring attention to the novel findings in the isolation and characterization of MVs, and the therapeutic application of MVs in VC.

Matrix vesicles from dental follicle cells improve alveolar bone regeneration via activation of the PLC/PKC/MAPK pathway.

BACKGROUND: The regeneration of bone loss that occurs after periodontal diseases is a significant challenge in clinical dentistry. Extracellular vesicles (EVs)-based cell-free regenerative therapies represent a promising alternative for traditional treatments. Developmental biology suggests matrix vesicles (MVs), a subtype of EVs, contain mineralizing-related biomolecules and play an important role in osteogenesis. Thus, we explore the therapeutic benefits and expect to find an optimized strategy for MV application. METHODS: Healthy human dental follicle cells (DFCs) were cultured with the osteogenic medium to generate MVs. Media MVs (MMVs) were isolated from culture supernatant, and collagenase-released MVs (CRMVs) were acquired from collagenase-digested cell suspension. We compared the biological features of the two MVs and investigated their induction of cell proliferation, migration, mineralization, and the modulation of osteogenic genes expression. Furthermore, we investigated the long-term regenerative capacity of MMVs and CRMVs in an alveolar bone defect rat model. RESULTS: We found that both DFC-derived MMVs and CRMVs effectively improved the proliferation, migration, and osteogenic differentiation of DFCs. Notably, CRMVs showed better bone regeneration capabilities. Compared to MMVs, CRMVs-induced DFCs exhibited increased synthesis of osteogenic marker proteins including ALP, OCN, OPN, and MMP-2. In the treatment of murine alveolar bone defects, CRMV-loaded collagen scaffold brought more significant therapeutic outcomes with less unhealing areas and more mature bone tissues in comparison with MMVs and acquired the effects resembling DFCs-based treatment. Furthermore, the western blotting results demonstrated the activation of the PLC/PKC/MAPK pathway in CRMVs-induced DFCs, while this cascade was inhibited by MMVs. CONCLUSIONS: In summary, our findings revealed a novel cell-free regenerative therapy for repairing alveolar bone defects by specific MV subtypes and suggest that PLC/PKC/MAPK pathways contribute to MVs-mediated alveolar bone regeneration.

The functional role of soluble proteins acquired by extracellular vesicles.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanosized particles released by all cell types during physiological as well as pathophysiological processes to carry out diverse biological functions, including acting as sources of cellular dumping, signalosomes and mineralisation nanoreactors. The ability of EVs to perform specific biological functions is due to their biochemical machinery. Among the components of the EVs' biochemical machinery, surface proteins are of critical functional significance as they mediate the interactions of EVs with components of the extracellular milieu, the extracellular matrix and neighbouring cells. Surface proteins are thought to be native, that is, pre-assembled on the EVs' surface by the parent cells before the vesicles are released. However, numerous pieces of evidence have suggested that soluble proteins are acquired by the EVs' surface from the extracellular milieu and further modulate the biological functions of EVs during innate and adaptive immune responses, autoimmune disorders, complement activation, coagulation, viral infection and biomineralisation. Herein, we will describe the methods currently used to identify the EVs' surface proteins and discuss recent knowledge on the functional relevance of the soluble proteins acquired by EVs.

Fluorescence evidence of annexin A6 translocation across membrane in model matrix vesicles during apatite formation.

Matrix vesicles (MVs) are 100-300 nm spherical structures released by mineralization competent cells to initiate formation of apatite, the mineral component in bones. Among proteins present in MVs, annexin A6 (AnxA6) is thought to be ubiquitously distributed in the MVs' lumen, on the surface of the internal and external leaflets of the membrane and also inserted in the lipid bilayer. To determine the molecular mechanism(s) that lead to the different locations of AnxA6, we hypothesized the occurrence of a pH drop during the mineralization. Such a change would induce the AnxA6 protonation, which in turn, and because of its isoelectric point of 5.41, would change the protein hydrophobicity facilitating its insertion into the MVs' bilayer. The various distributions of AnxA6 are likely to disturb membrane phospholipid organization. To examine this possibility, we used fluorescein as pH reporter, and established that pH decreased inside MVs during apatite formation. Then, 4-(14-phenyldibenzo[a,c]phenazin-9(14H)-yl)-phenol, a vibration-induced emission fluorescent probe, was used as a reporter of changes in membrane organization occurring with the varying mode of AnxA6 binding. Proteoliposomes containing AnxA6 and 1,2-Dimyristoyl-sn-glycero-3phosphocholine (DMPC) or 1,2-Dimyristoyl-sn-glycero-3phosphocholine: 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (DMPC:DPPS 9:1), to mimic the external and internal MV membrane leaflet, respectively, served as biomimetic models to investigate the nature of AnxA6 binding. Addition of Anx6 to DMPC at pH 7.4 and 5.4, or DMPC:DPPS (9:1) at pH 7.4 induced a decrease in membrane fluidity, consistent with AnxA6 interactions with the bilayer surface. In contrast, AnxA6 addition to DMPC:DPPS (9:1) at pH 5.4 increased the fluidity of the membrane. This latest result was interpreted as reflecting the insertion of AnxA6 into the bilayer. Taken together, these findings point to a possible mechanism of AnxA6 translocation in MVs from the surface of the internal leaflet into the phospholipid bilayer stimulated upon acidification of the MVs' lumen during formation of apatite.