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 (Ca2+) and inorganic phosphate (Pi) within their lumen and forming a nucleation core (NC). After further sequestration of Ca2+ and Pi, 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. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca2+) and non-mineralizing (-Ca2+) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca2+. This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.

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. Increased mineralization was observed after exposing the supports containing the DMPA:TNAP LB films to solutions of phospho-substrates, thus evidencing the role of TNAP on the growth of calcium phosphates after immobilization. These coatings deposited on metallic supports can be potentially applied as osteoconductive materials, aiming at the optimization of bone-substitutes integration in vivo.

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. Phosphatases (orphan phosphatase 1, ectonucleotide pyrophosphatase/phosphodiesterase 1 and tissue-nonspecific alkaline phosphatase) play a crucial role in controlling the inorganic pyrophosphate/inorganic phosphate ratio that allows MV-mediated initiation of mineralization. The lipidic microenvironment can help in the nucleation process of first crystals and also plays a crucial physiological role in the function of MV-associated enzymes and transporters (type III sodium-dependent phosphate transporters, annexins and Na+/K+ ATPase). The whole process is mediated and regulated by the action of several molecules and steps, which make the process complex and highly regulated. Liposomes and proteoliposomes, as models of biological membranes, facilitate the understanding of lipid-protein interactions with emphasis on the properties of physicochemical and biochemical processes. In this review, we discuss the use of proteoliposomes as multiple protein carrier systems intended to mimic the various functions of MVs during the initiation and propagation of mineral growth in the course of biomineralization. We focus on studies applying biophysical tools to characterize the biomimetic models in order to gain an understanding of the importance of lipid-protein and lipid-lipid interfaces throughout the process.

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. To this end, proteoliposomes were synthesized, composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS), with inserted tissue-nonspecific alkaline phosphatase (TNAP) and/or annexin V (AnxA5), both characteristic proteins of osteoblast-derived MVs. We then aimed to study how TNAP and AnxA5 insertion affects the proteoliposomes' membrane properties and, in turn, interactions with type II collagen, thus mimicking early MV activity during biomineralization. AFM images of these proteoliposomes, acquired in dynamic mode, revealed the presence of surface protrusions with distinct viscoelasticity, thus suggesting that the presence of the proteins induced local changes in membrane fluidity. Surface protrusions were measurable in TNAP-proteoliposomes but barely detectable in AnxA5-proteoliposomes. More complex surface structures were observed for proteoliposomes harboring both TNAP and AnxA5 concomitantly, resulting in a lower affinity for type II collagen fibers compared to proteoliposomes harboring AnxA5 alone. The present study achieved the topographic analysis of lipid vesicles by direct visualization of structural changes, resulting from protein incorporation, without the need for fluorescent probes.

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.