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 (Pi), a calcification promoter, and pyrophosphate (PPi), a calcification inhibitor. The Pi/PPi 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 PPi as well as Pi from ATP and tissue-nonspecific alkaline phosphatase (TNAP) that hydrolyzes both ATP and PPi to generate Pi. However, if and how these enzymes act in concert in MVs are still unclear. Herein, we investigated the role of NPP1 and TNAP in ATP hydrolysis during MV-mediated biomineralization using proteoliposomes as a biomimetic model for MVs. Proteoliposomes composed by 1,2-dipalmitoylphosphatidylcholine (DPPC) and harboring NPP1 alone, TNAP alone, or both together at different molar ratios (1:1, 10:1, and 1:10) were fabricated. After 48 h of incubation with ATP, TNAP-containing proteoliposomes consumed more ATP than NPP1-containing vesicles (270 and 210 nmol, respectively). Both types of vesicles comparatively formed ADP (205 and 201 nmol, respectively), while NPP1-containing vesicles hydrolyzed AMP less efficiently than TNAP-containing proteoliposomes (10 and 25 nmol, respectively). In vitro mineralization assays showed that in the presence of ATP, TNAP-harboring proteoliposomes mineralized through a sigmoidal single-step process, while NPP1-harboring vesicles displayed a two-step mineralization process. ATR-FTIR analyses showed that the minerals produced by TNAP-harboring proteoliposomes were structurally more similar to hydroxyapatite than those produced by NPP1-harboring vesicles. Our results with proteoliposomes indicate that the pyrophosphohydrolase function of NPP1 and the phosphohydrolase activity of TNAP act synergistically to produce a Pi/PPi ratio conducive to mineralization and the synergism is maximal when the two enzymes are present at equimolar concentrations. The significance of these findings for hypophosphatasia is discussed.

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. However, little is known about the mechanisms by which MVs can trigger this process. Here, we present a combination of in situ investigations and ex vivo analysis of MVs extracted from growing-femurs of chicken embryos to investigate the role played by phosphatidylserine (PS) in the formation of mineral nuclei. By using self-assembled Langmuir monolayers, we reconstructed the nucleation core - a PS-enriched motif thought to trigger mineral formation in the lumen of MVs. In situ infrared spectroscopy of Langmuir monolayers and ex situ analysis by transmission electron microscopy evidenced that mineralization was achieved in supersaturated solutions only when PS was present. PS nucleated amorphous calcium phosphate that converted into biomimetic apatite. By using monolayers containing lipids extracted from native MVs, mineral formation was also evidenced in a manner that resembles the artificial PS-enriched monolayers. PS-enrichment in lipid monolayers creates nanodomains for local increase of supersaturation, leading to the nucleation of ACP at the interface through a multistep process. We posited that PS-mediated nucleation could be a predominant mechanism to produce the very first mineral nuclei during MV-driven bone/cartilage biomineralization.

Synthesis of Sr-morin complex and its in vitro response: decrease in osteoclast differentiation while sustaining osteoblast mineralization ability.

Strontium ranelate (SrR) has been used as the ultimate choice for osteoporosis treatment. However, the development of more tolerable and bioactive Sr2+ carriers is still a need. The design of Sr2+-based platforms has moved towards the obtention of anion carriers that can also exhibit a positive effect on bone metabolism. In this sense, we used morin, a natural flavonoid, as a new arrangement for Sr2+ carriage in the synthesis of an Sr2+ complex. It has been claimed that phenolic compounds promote bone health. Therefore, we hypothesized that the association of Sr2+ with morin could improve its anabolic effects. Complexes with the general formula [(C15H9O7)Sr(H2O)2]Cl·3H2O were synthesized and characterized by elemental analysis, thermogravimetry, UV-Vis and infrared absorption spectroscopies and 1H-nuclear magnetic resonance. We showed that the complexation between morin and Sr2+ occurred among the 3-OH and 4C[double bond, length as m-dash]O groups of morin. Preosteoclasts cultures with the Sr-morin complex exhibited a reduced osteoclast differentiation rate and sustained osteoblast mineralization ability. The response of Sr-morin was higher than that observed for SrR at the same concentration range. Considering the above-mentioned observations, the Sr-morin complex could be an interesting approach to be further exploited not only as an alternative treatment for osteoporosis but also in the design of materials for faster osteointegration.

Different compact hybrid Langmuir-Blodgett-film coatings modify biomineralization and the ability of osteoblasts to grow.

Calcium phosphates (CaPs) are biomaterials widely used in tissue regeneration with outstanding biological performance. Although the tremendous improvements achieved in CaP's materials research over the years, their interaction with physiological environments still need to be fully understood. The aim of this study is to explore a biomimetic Langmuir-Blodgett (LB) membrane to template the growth of hydroxyapatite (HAp) coatings on Ti surfaces and the ability of these coatings in inducing biomineralization by osteoblasts cultured in vitro. Changing the phospholipids (i.e., dihexadecyl phosphate (DHP) or octadecylphosphonic acid (OPA)), we also tuned the surface Ca2+ concentration. This structural feature gave rise to different LB-hybrid surfaces where the concentration of Ca2+ in the OPA/HAp was higher than the concentration of Ca2+ in DHP/HAp coating. The higher Ca2+ amount on OPA/HAp coatings, allied to the physical-chemical features, lead to different responses on osteoblasts, stimulating or inhibiting the natural biomineralization. The OPA/HAp coating caused a delay in the osteoblast proliferation as indicated by the decrease in the cell viability at the 7th culture day. Improved cell differentiation triggered by the DHP/HAp coating resulted in higher osteoblast biomineralization. The present data underscore that besides both coatings being composed by HAp, the final interfacial composition and physical-chemical properties influence differently the osteoblast behavior. Although the best osteoblast's viability was found to OPA/HAp, our dataset attested that DHP/HAp induced mineralization more effectively than that. This unexpected finding highlight the importance of deeply understanding the biomaterial interface and suggest a promising approach to the design of biofunctional LB-based coatings with tunable properties. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2524-2534, 2018.

The role played by modified bioinspired surfaces in interfacial properties of biomaterials.

The success of a biomaterial relies on an appropriate interaction between the surface of that biomaterial and the surrounding environment; more specifically, the success of a biomaterial depends on how fluids, proteins, and cells interact with the foreign material. For this reason, the surface properties of biomaterial, such as composition, charge, wettability, and roughness, must be optimized for a desired application to be achieved. In this review we highlight different bioinspired approaches that are used to manipulate and fine-tune the interfacial properties of biomaterials. Inspired by noteworthy natural processes, researchers have developed materials with a functional anatomy that range from hierarchical hybrid structures to self-cleaning interfaces. In this review we focus on (1) the creation of particles and modified surfaces inspired by the structure and composition of biogenic mineralized tissues, (2) the development of biofunctional coatings, (3) materials inspired by biomembranes and proteins, and (4) the design of superwettable materials. Our intention is to point out different bioinspired methodologies that have been used to design materials for biomedical applications and to discuss how interfacial properties modified by manipulation of these materials determine their final biological response. Our objective is to present future research directions and to highlight the potential of bioinspired materials. We hope this review will provide an understanding of the interplay between interfacial properties and biological response so that successful biomaterials can be achieved.

Biomedical applications of nanotechnology.

The ability to investigate substances at the molecular level has boosted the search for materials with outstanding properties for use in medicine. The application of these novel materials has generated the new research field of nanobiotechnology, which plays a central role in disease diagnosis, drug design and delivery, and implants. In this review, we provide an overview of the use of metallic and metal oxide nanoparticles, carbon-nanotubes, liposomes, and nanopatterned flat surfaces for specific biomedical applications. The chemical and physical properties of the surface of these materials allow their use in diagnosis, biosensing and bioimaging devices, drug delivery systems, and bone substitute implants. The toxicology of these particles is also discussed in the light of a new field referred to as nanotoxicology that studies the surface effects emerging from nanostructured materials.

Biomimetic collagen/phospholipid coatings improve formation of hydroxyapatite nanoparticles on titanium.

Osseointegration between the surface of a certain material and the host tissue helps to evaluate the potential use of biomaterials in bone replacement. The physicochemical properties and biochemical composition of the material's surface regulates osseointegration. This study investigates how collagen into biomimetic matrixes affects hydroxyapatite (HAp) growth. Collagen was inserted into insoluble Langmuir monolayers containing either 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) or octadecylphosphonic acid (OPA) and transferred to titanium (Ti) supports by means of the Langmuir-Blodgett (LB) technique. The resulting films served as matrixes for HAp growth upon exposure of Ti discs to SBF. Scanning electron microscopy, atomic force microscopy, vibrational spectroscopy in the infrared region, X-ray diffraction, and energy dispersive X-ray spectroscopy aided characterization of the samples. Properties such as wettability, roughness, and surface free energy were also studied. The biocompatibility of the samples was investigated by osteoblast viability assays in vitro. Collagen interacted with DPPC and OPA at the air/water interface as evidenced by the pressure surface isotherms and the compressional modulus. Moreover, collagen in the subphase increased the stability of the phospholipid monolayer at high organization degree. Collagen incorporation into DPPC LB films induced formation of biomimetic HAp nanoparticles that resembled the HAp nanoparticles found in natural bone. Enhanced cell proliferation on the modified-Ti surfaces demonstrated that the coatings were not toxic to osteoblasts. These materials are potential candidates for bone-replacement applications.

Formation of carbonated hydroxyapatite films on metallic surfaces using dihexadecyl phosphate-LB film as template.

Hydroxyapatite serves as a bioactive material for biomedical purposes, because it shares similarities with the inorganic part of the bone. However, how this material deposits on metallic surfaces using biomimetic matrices remains unclear. In this study, we deposited dihexadecyl phosphate, a phospholipid that bears a simple chemical structure, on stainless steel and titanium surfaces using the Langmuir-Blodgett (LB) technique; we employed the resulting matrix to grow carbonated hydroxyapatite. We obtained the calcium phosphate coating via a two-step process: we immersed the surfaces modified with the LB films into phosphate buffer, and then, we exposed the metal to a solution that simulated the concentration of ions in the human plasma. The latter step generated carbonated hydroxyapatite, the same mineral existing in the bone. The free energy related to the surface roughness and composition increased after we modified the supports. We investigated the film morphology by scanning electron and atomic force microscopies and determined surface composition by infrared spectroscopy and energy dispersive X-ray. We also studied the role of the surface roughness and the surface chemistry on cell viability. The surface-modified Ti significantly increased osteoblastic cells proliferation, supporting the potential use of these surfaces as osteogenic materials.