Structural and biochemical characterization of a new type of lectin isolated from carp eggs.

A previously unidentified glycoprotein present in the eggs of the carp (Cyprinus carpio) was isolated and structurally characterized. The protein binds to a Sepharose 4B matrix and can be eluted with 0.4 M N-acetylglucosamine. The protein has an apparent molecular mass of 26686.3 Da. On the basis of gel-filtration chromatography, the protein appears to be present in solution as a monomer. The sequence of its 238 amino acids, the position of its four disulphide bridges and the composition of its single N-linked carbohydrate chain were determined. The lectin shows a very low agglutinating activity for human A-type erythrocytes and interacts with both Gram-positive and -negative bacteria. These latter interactions are inhibited by N-acetylglucosamine. A database search shows that its amino acid sequence is similar to that of the members of an invertebrate lectin family that includes tachylectin-1. Tachylectin-1 is present in the amoebocytes of the horseshoe crab, Tachypleus tridentatus, and plays a role in the innate defence system of this species. Homologous genes are also present in other fish, having 85% identity with a gene expressed in the oocytes of the crucian carp (Carassius auratus gibelio) and 78% identity with a gene in the cDNA library of the zebrafish (Danio rerio)

FbsA-driven fibrinogen polymerization: a bacterial Deceiving Strategy.

We show that FbsA, a cell wall protein of the bacterium Streptococcus agalactiae, promotes large-scale aggregation of human plasma fibrinogen, leading to the formation of a semiflexible polymerlike network. This extensive aggregation process takes place not only in solution, but also on FbsA-functionalized colloidal particles, and leads to the formation of a thick layer on the bacterial cell wall itself, which becomes an efficient mask against phagocytosis

In Vitro Production of Calcified Bone Matrix onto Wool Keratin Scaffolds via Osteogenic Factors and Electromagnetic Stimulus

Pulsed electromagnetic field (PEMF) has drawn attention as a potential tool to improve the ability of bone biomaterials to integrate into the surrounding tissue. We investigated the effects of PEMF (frequency, 75 Hz; magnetic induction amplitude, 2 mT; pulse duration, 1.3 ms) on human osteoblast-like cells (SAOS-2) seeded onto wool keratin scaffolds in terms of proliferation, differentiation, and production of the calcified bone extracellular matrix. The wool keratin scaffold offered a 3D porous architecture for cell guesting and nutrient diffusion, suggesting its possible use as a filler to repair bone defects. Here, the combined approach of applying a daily PEMF exposure with additional osteogenic factors stimulated the cells to increase both the deposition of bone-related proteins and calcified matrix onto the wool keratin scaffolds. Also, the presence of SAOS-2 cells, or PEMF, or osteogenic factors did not influence the compression behavior or the resilience of keratin scaffolds in wet conditions. Besides, ageing tests revealed that wool keratin scaffolds were very stable and showed a lower degradation rate compared to commercial collagen sponges. It is for these reasons that this tissue engineering strategy, which improves the osteointegration properties of the wool keratin scaffold, may have a promising application for long term support of bone formation in vivo

Calcium-phosphate glass-based bioresorbable fibre optics for light and drug delivery

Calcium-phosphate glasses (CPGs) are commonly used as scaffolds in tissue engineering. A novel formulation of optically transparent CPG has been recently developed to be used as an optical fibre for biomedical implantable devices. Its purpose is to combine the bioresorbability of CPGs with optical features, thus extending the applications of bioresorbable sensors for in-body monitoring or diagnostics. Modifications of the glass composition or post-treatments on the fibres can tailor the dissolution time and the interaction of the glass with different stimuli as well as with specific cells. The tested glasses both in bulk and fibre shapes showed good strength (from 200 to 350 MPa) with values that are lower than standard silica glass and much higher than common bioresorbable polymers. CPG fibres were also implanted in living rats for several weeks and no clinical signs of any adverse effect have been found. We will present our latest results on these subjects starting from the characterisation of the CPGs by means of dissolution tests, in-vitro, and ex-vivo experiments

An in vivo Comparison Study Between Strontium Nanoparticles and rhBMP2

The osteoinductive property of strontium was repeatedly proven in the last decades. Compelling in vitro data demonstrated that strontium hydroxyapatite nanoparticles exert a dual action, by promoting osteoblasts-driven matrix secretion and inhibiting osteoclasts-driven matrix resorption. Recombinant human bone morphogenetic protein 2 (rhBMP2) is a powerful osteoinductive biologic, used for the treatment of vertebral fractures and critically-sized bone defects. Although effective, the use of rhBMP2 has limitations due its recombinant morphogen nature. In this study, we examined the comparison between two osteoinductive agents: rhBMP2 and the innovative strontium-substituted hydroxyapatite nanoparticles. To test their effectiveness, we independently loaded Gelfoam sponges with the two osteoinductive agents and used the sponges as agent-carriers. Gelfoam are FDA-approved biodegradable medical devices used as delivery system for musculoskeletal defects. Their porous structure and spongy morphology make them attractive in orthopedic field. The abiotic characterization of the loaded sponges, involving ion release pattern and structure investigation, was followed by in vivo implantation onto the periosteum of healthy mice and comparison of the effects induced by each implant was performed. Abiotic analysis demonstrated that strontium was continuously released from the sponges over 28 days with a pattern similar to rhBMP2. Histological observations and gene expression analysis showed stronger endochondral ossification elicited by strontium compared to rhBMP2. Osteoclast activity was more inhibited by strontium than by rhBMP2. These results demonstrated the use of sponges loaded with strontium nanoparticles as potential bone grafts might provide better outcomes for complex fractures. Strontium nanoparticles are a novel and effective non-biologic treatment for bone injuries and can be used as novel powerful therapeutics for bone regeneration

Mesoporous bioactive glass as a multifunctional system for bone regeneration and controlled drug release

Purpose: Coupling the potential for bone regeneration and the ability for in situ controlled drug release in a single device is a challenging field of research in bone tissue engineering; in an attempt to pursue this aim, mesoporous bioactive glass (MBG) membranes belonging to the SiO2-P2O5-CaO ternary system were produced and characterized. Methods: The glass was synthesized via a sol-gel route coupled with an evaporation-induced self-assembly process by using a non-ionic block co-polymer as a mesostructure former. MBG structure and morphology, as well as mesopores size and shape, were investigated by x-ray diffraction, transmission electron microscopy, and N2 adsorption-desorption measurements. In vitro bioactivity was investigated by soaking MBG membranes in simulated body fluid (SBF) for different time frames. Ibuprofen was encapsulated into MBG pores and drug release kinetics in SBF were assessed. Biological tests by using SAOS-2 cells were performed to assess the material cytocompatibility. Results: The material revealed significant ability to induce hydroxyapatite formation on its surface (bioactivity). Drug release kinetics in SBF are very similar to those obtained for mesoporous silica having mesopore size comparable to that of the prepared MBG (∼5 nm). No evidence of cell viability depression was detected during in vitro culture, which demonstrates the good biological compatibility of the material. Conclusions: The easiness of tailoring and shaping, the highly bioactive and biocompatible behavior, and the drug uptake/release ability of the prepared materials may suggest their use as "smart" multifunctional grafts for bone reconstructive surgery

Enhancement of the Biological and Mechanical Performances of Sintered Hydroxyapatite by Multiple Ions Doping

In the present work, hydroxyapatite (HA) nanoparticles doped with Mg2+, Sr2+, and Zn2+ ions are developed by wet neutralization method and then sintered at 1,250°C to obtain bulk consolidated materials. Physicochemical and microstructural analyses show that the presence of doping ions in the HA structure induced the formation of βTCP as secondary phase, during the sintering process, and we found that this effect is depending on the stability of the various doping ions in the hydroxyapatite lattice itself. We also found that the formation of βTCP as secondary phase, in turn, confines the grain growth of HA induced by the high-temperature sintering process, thus leading to a strong increase of the flexural strength of the bulk materials, according to Hall-Petch-like law. Furthermore, we found that the doping ions enter also in the structure of the βTCP phase; besides the grain growth confinement, also the solubility and ion release ability of the final materials were enhanced. In addition to ameliorate the mechanical performance, the described phenomena also activate multiple biofunctionalities: (i) ability to upregulate various genes involved in the osteogenesis, as obtained by human adipose stem cells culture and evaluated by array technology; (ii) enhanced resistance to the adhesion and proliferation of Gram+ and Gram– bacterial strains. Hence, our results open a perspective for the use of sintered multiple ion-doped HA to develop ceramic biodevices, such as plates, screws, or other osteosynthesis media, with enhanced strength, osteointegrability, and the ability to prevent post-surgical infections

Atmospheric pressure non-equilibriumplasma for the production of composite materials

In the evolving field of tissue engineering, continuous advances are required to improve scaffold design and fabrication to obtain biomimetic supports for cell adhesion, proliferation, penetration and differentiation. Both electrospun fibrous scaffolds and hydrogels are used in this field since they well reproduce the structure of the extracellular matrix (ECM) of many biological tissues. Limitations of these two types of materials can be overcome through their combination, by developing composite structures combining enhanced mechanical properties (provided by the fibrous components) and improved cell penetration (provided by the gel phase) in a superior ability to mimic natural ECM that is constituted by both a fibrous protein network and a hydrogel matrix. Here we develop new composite materials made of electrospun PLLA scaffolds and poly(amidoamine) hydrogels with different degrees of crosslinking. To promote compatibilization and good adhesion between the two materials, surface chemical reactions between hydrogels and PLLA mats are induced by inserting amino functional groups on electrospun PLLA mats by means of atmospheric pressure non-thermal plasma. Results will be presented concerning the exposure of PLLA substrates to the plasma region generated by a Dielectric Barrier Discharge at atmospheric pressure, driven by a HV Amplifier connected to a function generator operating with a microsecond rise time and operated in N2. Surface and solid-state thermo-mechanical characterizations of plasma treated substrates and of resulting composite materials at different crosslinking degrees are presented. Results of mechanical tests show a high adhesion between hydrogel and plasma treated PLLA electrospun mats, underlining the opportunity to use atmospheric non-thermal plasmas to fabricate a composite starting from two materials otherwise physically incompatible. Potential effects of nanofibrous-hydrogel were evaluated by investigating pluripotent stem cells response

The NATO project : nanoparticle-based countermeasures for microgravity-induced osteoporosis

Recent advances in nanotechnology applied to medicine and regenerative medicine have an enormous and unexploited potential for future space and terrestrial medical applications. The Nanoparticles and Osteoporosis (NATO) project aimed to develop innovative countermeasures for secondary osteoporosis affecting astronauts after prolonged periods in space microgravity. Calcium- and Strontium-containing hydroxyapatite nanoparticles (nCa-HAP and nSr-HAP, respectively) were previously developed and chemically characterized. This study constitutes the first investigation of the effect of the exogenous addition of nCa-HAP and nSr-HAP on bone remodeling in gravity (1\u2009g), Random Positioning Machine (RPM) and onboard International Space Station (ISS) using human bone marrow mesenchymal stem cells (hBMMSCs). In 1\u2009g conditions, nSr-HAP accelerated and improved the commitment of cells to differentiate towards osteoblasts, as shown by the augmented alkaline phosphatase (ALP) activity and the up-regulation of the expression of bone marker genes, supporting the increased extracellular bone matrix deposition and mineralization. The nSr-HAP treatment exerted a protective effect on the microgravity-induced reduction of ALP activity in RPM samples, and a promoting effect on the deposition of hydroxyapatite crystals in either ISS or 1\u2009g samples. The results indicate the exogenous addition of nSr-HAP could be potentially used to deliver Sr to bone tissue and promote its regeneration, as component of bone substitute synthetic materials and additive for pharmaceutical preparation or food supplementary for systemic distribution

Heterogeneous and self-organizing mineralization of bone matrix promoted by hydroxyapatite nanoparticles

The mineralization process is crucial to the load-bearing characteristics of the bone extracellular matrix. In this work, we have studied the spatiotemporal dynamics of mineral deposition by human bone marrow mesenchymal stem cells differentiating toward osteoblasts promoted by the presence of exogenous hydroxyapatite nanoparticles. At molecular level, the added nanoparticles positively modulated the expression of bone-specific markers and enhanced calcified matrix deposition during osteogenic differentiation. The nucleation, growth and spatial arrangement of newly deposited hydroxyapatite nanocrystals have been evaluated using Scanning Micro X-Ray Diffraction and Scanning Micro X-Ray Fluorescence. As leading results, we have found the emergence of a complex scenario where the spatial organization and temporal evolution of the process exhibit a heterogeneous and self-organizing dynamics. At the same time the possibility to control the differentiation kinetic through the addition of synthetic nanoparticles, paves the way to empower the generation of more structured bone scaffolds in tissue engineering and to design new drugs in regenerative medicine