A protein corona modulates the function of mineralization-competent matrix vesicles.

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.

Differential effects of the lipidic and ionic microenvironment on NPP1's phosphohydrolase and phosphodiesterase activities.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an enzyme present in matrix vesicles (MV). NPP1 participates on the regulation of bone formation by producing pyrophosphate (PP) from adenosine triphosphate (ATP). Here, we have used liposomes bearing dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), and cholesterol (Chol) harboring NPP1 to mimic the composition of MV lipid rafts to investigate ionic and lipidic influence on NPP1 activity and mineral propagation.

Annexin A5 stabilizes matrix vesicle-biomimetic lipid membranes: unravelling a new role of annexins in calcification.

Matrix vesicles are a special class of extracellular vesicles thought to actively contribute to both physiologic and pathologic mineralization. Proteomic studies have shown that matrix vesicles possess high amounts of annexin A5, suggesting that the protein might have multiple roles at the sites of calcification. Currently, Annexin A5 is thought to promote the nucleation of apatitic minerals close to the inner leaflet of the matrix vesicles' membrane enriched in phosphatidylserine and Ca.

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization.

Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (P) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos.

Trypanosoma cruzi nitroreductase: Structural features and interaction with biological membranes.

Due to its severe burden and geographic distribution, Chagas disease (CD) has a significant social and economic impact on low-income countries. Benznidazole and nifurtimox are currently the only drugs available for CD. These are prodrugs activated by reducing the nitro group, a reaction catalyzed by nitroreductase type I enzyme from Trypanosoma cruzi (TcNTR), with no homolog in the human host.

Ultrasensitive Diamond Microelectrode Application in the Detection of Ca Transport by AnnexinA5-Containing Nanostructured Liposomes.

This report describes the innovative application of high sensitivity Boron-doped nanocrystalline diamond microelectrodes for tracking small changes in Ca concentration due to binding to Annexin-A5 inserted into the lipid bilayer of liposomes (proteoliposomes), which could not be assessed using common Ca selective electrodes. Dispensing proteoliposomes to an electrolyte containing 1 mM Ca resulted in a potential jump that decreased with time, reaching the baseline level after ~300 s, suggesting that Ca ions were incorporated into the vesicle compartment and were no longer detected by the microelectrode. This behavior was not observed when liposomes (vesicles without AnxA5) were dispensed in the presence of Ca.

NPP1 and TNAP hydrolyze ATP synergistically during biomineralization.

Matrix vesicles (MVs) are a special class of extracellular vesicles released by mineralizing cells during bone and tooth mineralization that initiate the precipitation of apatitic minerals by regulating the extracellular ratio between inorganic phosphate (P), a calcification promoter, and pyrophosphate (PP), a calcification inhibitor. The P/PP ratio is thought to be controlled by two ecto-phosphatases present on the outer leaflet of the MVs' membrane: ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) that produces PP as well as P from ATP and tissue-nonspecific alkaline phosphatase (TNAP) that hydrolyzes both ATP and PP to generate P. However, if and how these enzymes act in concert in MVs are still unclear.

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.

Phosphatidylserine controls calcium phosphate nucleation and growth on lipid monolayers: A physicochemical understanding of matrix vesicle-driven biomineralization.

Bone biomineralization is an exquisite process by which a hierarchically organized mineral matrix is formed. Growing evidence has uncovered the involvement of one class of extracellular vesicles, named matrix vesicles (MVs), in the formation and delivery of the first mineral nuclei to direct collagen mineralization. MVs are released by mineralization-competent cells equipped with a specific biochemical machinery to initiate mineral formation.

Characterization of the in Vitro Osteogenic Response to Submicron TiO Particles of Varying Structure and Crystallinity.

Titanium oxide (TiO) nano-/microparticles have been widely used in orthopedic and dental sciences because of their excellent mechanical properties, chemical stability, and ability to promote the osseointegration of implants. However, how the structure and crystallinity of TiO particles may affect their osteogenic activity remains elusive. Herein, we evaluated the osteogenic response to submicron amorphous, anatase, and rutile TiO particles with controlled size and morphology.

Matrix vesicle biomimetics harboring Annexin A5 and alkaline phosphatase bind to the native collagen matrix produced by mineralizing vascular smooth muscle cells.

Backgound: Vascular smooth muscle cells (VSMCs) transdifferentiated ectopically trigger vascular calcifications, contributing to clinical cardiovascular disease in the aging population. AnxA5 and TNAP play a crucial role in (patho)physiological mineralization.

Methods: We performed affinity studies between DPPC and 9:1 DPPC:DPPS-proteoliposomes carrying AnxA5 and/or TNAP and different types of collagen matrix: type I, II, I + III and native collagenous extracellular matrix (ECM) produced from VSMCs with or without differentiation, to simulate ectopic calcification conditions.

Entropy-driven binding of octyl gallate in albumin: Failure in the application of temperature effect to distinguish dynamic and static fluorescence quenching.

Fluorescence quenching is widely used to obtain association constants between proteins and ligands. This methodology is based on assumption that ground-state complex between protein and ligand is responsible for quenching. Here, we call the attention about the risk of using the temperature criterion for decision of applying or not fluorescence quenching data to measure association constants.

Quantitative atomic force microscopy provides new insight into matrix vesicle mineralization.

Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca) and inorganic phosphate (P) within their lumen and forming a nucleation core (NC). After further sequestration of Ca and P, the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples.

Is alkaline phosphatase biomimeticaly immobilized on titanium able to propagate the biomineralization process?

Tissue-nonspecific alkaline phosphatase (TNAP) is a key enzyme in the biomineralization process as it produces phosphate from a number of phospho-substrates stimulating mineralization while it also inactivates inorganic pyrophosphate, a potent mineralization inhibitor. We have previously reported on the reconstitution of TNAP on Langmuir monolayers as well as proteoliposomes. In the present study, thin films composed of dimyristoylphosphatidic acid (DMPA) were deposited on titanium supports by the Langmuir-Blodgett (LB) technique, and we determined preservation of TNAP's phosphohydrolytic activity after incorporation into the LB films.

Lipid microenvironment affects the ability of proteoliposomes harboring TNAP to induce mineralization without nucleators.

Tissue-nonspecific alkaline phosphatase (TNAP), a glycosylphosphatidylinositol-anchored ectoenzyme present on the membrane of matrix vesicles (MVs), hydrolyzes the mineralization inhibitor inorganic pyrophosphate as well as ATP to generate the inorganic phosphate needed for apatite formation. Herein, we used proteoliposomes harboring TNAP as MV biomimetics with or without nucleators of mineral formation (amorphous calcium phosphate and complexes with phosphatidylserine) to assess the role of the MVs' membrane lipid composition on TNAP activity by means of turbidity assay and FTIR analysis. We found that TNAP-proteoliposomes have the ability to induce mineralization even in the absence of mineral nucleators.

Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis, properties, functions and biomimetic models.

Background: Matrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100-300nm diameter harboring the biochemical machinery needed to induce mineralization.

Biophysical aspects of biomineralization.

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix (ECM) by promoting the synthesis of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Several lipid and proteins present in the membrane of the MVs mediate the interactions of MVs with the ECM and regulate the initial mineral deposition and posterior propagation. Among the proteins of MV membranes, ion transporters control the availability of phosphate and calcium needed for initial HA deposition.

Topographic analysis by atomic force microscopy of proteoliposomes matrix vesicle mimetics harboring TNAP and AnxA5.

Atomic force microscopy (AFM) is one of the most commonly used scanning probe microscopy techniques for nanoscale imaging and characterization of lipid-based particles. However, obtaining images of such particles using AFM is still a challenge. The present study extends the capabilities of AFM to the characterization of proteoliposomes, a special class of liposomes composed of lipids and proteins, mimicking matrix vesicles (MVs) involved in the biomineralization process.

Pendant-drop method coupled to ultraviolet-visible spectroscopy: A useful tool to investigate interfacial phenomena.

UV-vis spectroscopy is a powerful tool to investigate surface phenomena. Surface tension measurements coupled to spectroscopic techniques can help to elucidate how the interface organization influences the electronic properties of molecules. However, appreciable sample volumes are usually necessary to achieve strong signals during conduction of experiments.

Liposomal systems as carriers for bioactive compounds.

Since the revolutionary discovery that phospholipids can form closed bilayered structures in aqueous systems, the study of liposomes has become a very interesting area of research. The versatility and amazing biocompatibility of liposomes has resulted in their wide-spread use in many scientific fields, and many of their applications, especially in medicine, have yielded breakthroughs in recent decades. Specifically, their easy preparation and various structural aspects have given rise to broadly usable methodologies to internalize different compounds, with either lipophilic or hydrophilic properties.

Proteoliposomes with the ability to transport Ca(2+) into the vesicles and hydrolyze phosphosubstrates on their surface.

We describe the production of stable DPPC and DPPC:DPPS-proteoliposomes harboring annexin V (AnxA5) and tissue-nonspecific alkaline phosphatase (TNAP) and their use to investigate whether the presence of AnxA5 impacts the kinetic parameters for hydrolysis of TNAP substrates at physiological pH. The best catalytic efficiency was achieved in DPPS 10%-proteoliposomes (molar ratio), conditions that also increased the specificity of TNAP hydrolysis of PPi. Melting behavior of liposomes and proteoliposomes was analyzed via differential scanning calorimetry.

Catalytic signature of a heat-stable, chimeric human alkaline phosphatase with therapeutic potential.

Recombinant alkaline phosphatases are becoming promising protein therapeutics to prevent skeletal mineralization defects, inflammatory bowel diseases, and treat acute kidney injury. By substituting the flexible crown domain of human intestinal alkaline phosphatase (IAP) with that of the human placental isozyme (PLAP) we generated a chimeric enzyme (ChimAP) that retains the structural folding of IAP, but displays greatly increased stability, active site Zn²⁺ binding, increased transphosphorylation, a higher turnover number and narrower substrate specificity, with comparable selectivity for bacterial lipopolysaccharide (LPS), than the parent IAP isozyme. ChimAP shows promise as a protein therapeutic for indications such as inflammatory bowel diseases, gut dysbioses and acute kidney injury.