Biomimetic bioactive biomaterials – the next generation of implantable devices

Resume : The effective regeneration of fully functional tissues which contains bone structures is one of the primary challenges in regenerative medicine and a major challenge for reconstructive and orthopedic surgery that repairing of bone defects. Currently, the main approaches are surgical reconstruction using autografts or allografts which are convenient but contain some limitations associating with the availability of autografts, the risk of immunogenicity and infection. Undoubtedly, developing biomaterial scaffolds that can mechanically and functionally support and assist in bone regeneration is required. In this report, continuous poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) and PHBV/polyaspartic acid (PAA) nanofibers with high porosity, high ratio of surface area to mass and superior mechanical properties were fabricated through electrospinning technic. Despite good mechanical properties and predictable biodegradation kinetics, PHBV and PHBV/PAA nanofibers can not provide a favorable surface for cell adhesion and proliferation due to lack of specific cell-recognition moieties. We employed a calcium-phosphate dipping method to deposit the nano-hydroxyapatite (nHA) on the nanofibers to obtain desired PHBV-nHA and PHBV/PAA-nHA scaffolds with the property of osteoconductivity. Field emission scanning electron microscope (FE-SEM), Fourier transform infrared spectroscopy (FTIR), and tensile tester were employed to investigate the mechanical properties of PHBV, PHBV-nHA, PHBV/PAA and PHBV/PAA-nHA nanofibers. Human fetal osteoblast cells were cultured on these scaffolds for evaluating the cell proliferation and mineralization. Morphological evaluation on firmed the fiber diameters of PHBV and PHBV/PAA as 447±69 nm and 368±124 nm, respectively. There was no obvious changes about the diameters after depositing nHA. However, the surface of the scaffold transformed from hydrophobic to hydrophilic after the deposition of nHA with water contact angles of 129.4°?114.2°?82.8° and 51.7° for PHBV, PHBV/PAA, PHBV/PAA-nHA and PHBV/PAA-nHA nanofibers. Initial adhesion of hFOB is especially critical for long-term stability and mineralization of the cells; thus, the capacity for nanofiber scaffolds to support hFOB adhesion and proliferation was evaluated using cell proliferation assay at day 5, 10 and 15. It was obvious that the cells on all of the scaffolds kept increasing during the process of culture. Especially, cells on PHBV-nHA scaffold had 35.10% higher proliferation than that on pure PHBV scaffold. Comparing with PHBV/PAA nanofibers, cell on PHBV/PAA-nHA nanofibers had a higher proliferation of 26.68%. The biological advantages of adding nHA are crucial since it is the major inorganic component of the bone matrix and it has specific affinity towards many adhesive proteins. The CMFDA (5-chloromethylfluorescein diacetate) images on day 15 showed that the cells on nanofiber scaffolds formed noticeably more colonies than that on control surfaces, owing to the favorable nanofibrous architecture which promoted cellular activities. Evidence suggesting cellular infiltration was observed via FE-SEM, multi-layers of cells on all scaffolds with mineral particles on the surface indicate confluent cell growth with cell-secreted mineralization. By day 15, high mineralization was observed with fused cells forming a thick layer on the surface of the scaffolds along with the cell ECM deposits. Notably, PHBV/PAA-nHA scaffolds were apparently in favor of producing more mineral nodules and appeared to aggregate and coalesce into large mineral clumps compared to other scaffolds. nHA initiated an increase in osteoblast adhesion, osteo-integration and deposition of calcium containing minerals on the surface of the scaffolds, thus enhancing new bone formation. Nanofibrous scaffolds contained nHA had the ability to accelerate the growth and maturity of hFOB cells. The observed results proved that the PHBV/PAA-nHA scaffolds promoted greater osteogenic mineralization of hFOB as evident from the enzyme activity and mineralization profiles for bone tissue engineering.

Resume : In this work, we show that self-assembled heparin-mimetic peptide nanofiber gel is an effective bioactive wound dressing for the healing of full-thickness excisional wounds in the rat model. The bioactive gel treated wounds exhibited increased re-epithelization, skin appendage formation and granulation tissue organization compared to sucrose-treated samples. Increased blood vessel numbers in the gel-treated wounds at day 7 suggest that angiogenesis plays a key role in improvement in tissue healing for bioactive gel-treated wounds. The heparin-mimetic peptide nanofiber gel is a promising platform for enhancing the scar-free recovery of acute wounds. Wound repair in adult mammals typically ends with the formation of a scar, which prevents full restoration of the function of the healthy tissue. Rapid and natural recovery of the major wound injuries require development of therapeutic approaches that can enhance the healing process including mechanical and biochemical problems.

Resume : Introduction There is growing evidence that excessive reactive oxygen species (ROS) are associated with intervertebral disc degeneration (IVD). High levels of ROS inhibit proliferation, induce premature senescense and promote catabolic phenotype of nucleus pulposus (NP) cells1. Thus, development of biomaterial systems that efficiently regulate ROS levels have been considered to promote disc regeneration. Herein, antioxidant hollow polymer microreactors were prepared and their potential to prevent oxidative stress and modulate extracellular matrix activity in interleukin-1? (IL-1?) induced inflammation model of NP was evaluated. Materials & Methods Hollow polymer microreactors were fabricated via the layer-by-layer approach employing poly(allylamine hydrochloride), dextran and tannic acid (TA) as polyelectrolytes and catalase-loaded CaCO3 microparticles as sacrificial templates. These microreactors were fully characterized in terms of morphology, physico-chemical properties and H2O2 scavenging capacity. NP cell viability and metabolic activity in the presence of these microreactors was analyzed by Live/Dead® and AlamarBlue® assays, respectively. Oxidative stress in NP cells stimulated with IL-1?2 was evaluated with CellRox® fluorogenic probes under confocal microscopy. The mRNA expression of catabolic enzymes of MMP-3 and ADAMTS-5, and matrix component of aggrecan and collagen II were also determined by RT-qPCR. Results & Discussion Catalase was satisfactorily incorporated into CaCO3 microparticles (2.6±0.6 ?m) via the coprecipitation method. After polyelectrolyte deposition and subsequent template removal by EDTA, hollow polymer microreactors encapsulating catalase were obtained. These microreactors were able to scavenge H2O2 in a concentration-dependent manner. As an illustration, H2O2 concentration dropped from 10 to 8 or 4?M upon the reaction with 100 or 400 ?g/ml of microreactors, respectively. In vitro, oxidative stress of IL-1? stimulated NP cells was greatly reduced after treatment with hollow polymer microreactors. Conclusions Hollow polymer microreactors encapsulating catalase and with an external layer of tannic acid showed antioxidant properties and were able to prevent oxidative stress in inflamed NP cells. Acknowledgments This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) and is co-funded under the European Regional Development Fund under Grant Number 13/RC/2073. A.L. would like to acknowledge Basque Government (Department of Education, Language Policy and Culture) for a postdoctoral grant. References 1. Dimozi A, Mavrogonatou E, Sklirou A, Kletsas D. European Cells and Materials 2015; 30: 89-103. 2. Isa ILM, Srivastava A, Tiernan D, Owens P, Rooney P, Dockery P, Pandit A. Biomacromolecules 2015; 16: 1714-1725.

Resume : Introduction Artificial scaffolds mimicking the morphology, mechanical properties and biochemical complexity of natural tissues are of vital importance for tissue engineering applications. Tendonous tissues are comprised of aligned collagen fibrils providing topographical guidance for cells and mechanical strength; however, an often overlooked physical property of collagenous tissues is an inherent piezoelectric response which is speculated as having a functional role in musculoskeletal homeostasis Interestingly, piezoelectric materials can exploit the loading capacity of tendon tissue to produce stimulating current which can be hypothesized is important for tissue repair. In this study, a piezoelectric fibrous scaffold was developed from Poly poly[(vinylidenefluoride-co-trifluoroethylene] P(VDF-TrFE), a material capable of generating electrical charges under mechanical loading in physiological conditions to maintain cell tenogenic phenotype. In addition, fiber alignment for adequate topographic guidance of the cells and piezoelectrical properties were controlled through the incorporation of Boron Nitride Nanotubes (BNNT) which have same structure as MWCNT but present piezoelectricity. Materials and Methods P(VDF-TrFE) was dissolved in DMF/Acetone and electrospun into fine fibers by changing the voltage, mandrel rotation speed and the distance between tip and collector with a constant flow rate. To improve the mechanical properties, BNNT were coated with amphiphilic polyvinyl pyrrolidone (PVP) using an ultrasonication process and incorporated into the matrix. The scaffold morphology was observed by FESEM and the electrical properties in response to cyclic loading were assessed with PFM and an in-house oscilloscope/dynamic loading system. Primary Human Tenocytes were isolated and cultured for 1, 3 ,7 and 14 days onto the scaffolds under static and mechanical loading conditions. Samples were, stained for focal adhesions (FA) using an anti-vinculin antibody. The number of FA and its length were counted using confocal microscopy and proliferation of cells was assessed via Alamar blue®. Gene expression was evaluated by RT-PCR using different markers such as Osterix, Osteocalcin, Collagen Type I and GAPDH. Results The results show that PDVF-TrFE/BNNT scaffolds are biocompatible and stimulate differentiation of Tenocytes cells; and attachment and growth of fibroblasts on scaffolds. The scaffold cytocompatibility was evaluated by Alamar blue® assay and cells demonstrated cell viability for both nanohybrid scaffolds and pristine P(VDF-TrFE) meshes. Preliminary results shows that cell morpohology and focal adhesion distribution in cells cultured on composite scaffold were distinctly different from those on pristine scaffold. Tenocytes grown on the composite scaffolds exhibited a more elongated morphology while cells on pristine scaffolds were orientated randomly. Discussion and Conclusions This study indicates that the physical properties of piezoelectric PVDF-TrFE/BNNT fibers may be readily controlled by electrospinning and through the incorporation of BNNT to fabricate tailored piezoelectric scaffolds for biomedical applications. Acknowledgments Science Foundation Ireland: SIRG COFUND fellowship (grant agreement no. 11/SIRG/B2135), Centre for Research in Medical Devices (CÚRAM) (Grant agreement no. 13/RC/2073)

Resume : Nanoparticle-based cancer theranostics represent an emerging and novel approach to cancer treatment. The unique properties of nanoparticles, such as small size, large surface area, multifunctional groups available on surface, facilitate their applications in cancer treatment compared to classical pharmaceutics. Cancer hyperthermia based on magnetic nanoparticles is one of the great advantageous treatment and overcomes drawbacks of established cancer therapies. It deals with thermal ablation of cancer cells at temperature between 42–45 oC preferentially eradicate them through an expeditive apoptotic cell death without damaging normal tissues. Unique advantages of hyperthermia using nanomaterials include spatiotemporally controlled treatments of targeted diseases in a noninvasive manner. However, cancer hyperthermia possesses extreme challenges including the diagnostic sensitivity, treatment efficacy, and bioavailability of nanoparticles as well as the heterogeneity and drug resistance of tumors for eventual clinical implementation. In our current project, we aim to introduce novel magnetic-platinum core-shell nanosystem as a tumor pH-sensitive drug carrier for effective cancer theranostics. The developed core-shell system consists of two subunits, first is magnetic (Fe3O4) core with platinum shell (Pt), Pt shell improves hyperthermia performance of magnetic nanoparticles. The second is surface modalities of core-shell system that can allow anticancer drug and deliver it into cancer cells with temperature and pH. The developed system can deliver drugs and overcome cancer drug resistance through combined effect of magnetic hyperthermia and controlled drug release. The two functions are designed to be triggered only by the heat generated through the application of an alternating magnetic field (AMF). We have successfully visualized cancer cells by using these core-shell nanostructures before and after hyperthermia via a T2 MR imaging. Cancer cells incubated with these core-shell nanoparticles are detected in MR imaging, demonstrating early stage diagnosis of tumors without using any targeting agents. Furthermore, combined hyperthermia and pH-triggered drug release enabled efficient hyperthermia to selectively kill cancer cells. In particular, we demonstrated the superior therapeutic efficacy of core shell nanostructure in highly heterogeneous drug-resistant tumors, showing a great potential for further clinical applications.

Resume : Among nanoantimicrobials [1], silver nanophases are widely employed as bioactive additives in industrial goods to inhibit the proliferation of several pathogen microorganisms. National and international health agencies, however, have questioned about the safety of these nanophases for humans and environment [2]. To this aim, in a recent project, we worked not only on the modification of polyurethane-based materials by photo-deposited silver nanoparticles (Ag-PU) [3] for applications in air-filtering systems, stuffing for seats, etc., but also on their detailed characterization, including nanosafety. In this communication, we present the analytical characterization of the composite material in terms of morphology (TEM, SEM, AFM), surface chemical composition (XPS), ionic release in contact media (ETAAS, ICP-MS), bioactivity, as well as whole nanoparticle release. After setting up a proper experimental protocol, the last aspect was investigated by single-particle-ICP-MS and TEM, aiming at quantifying the extent of whole particle release by antimicrobial foams under real-life usage conditions. The proposed systematic approach allows defining suitable composite final properties, in terms of bioactivity and safety, by proper tuning of deposition parameters. [1] N. Cioffi, M. Rai Eds, Nano-antimicrobials. Progress and Prospects, Springer-Verlag (2012). [2] T. Faunce, A. Watal, Nanomedicine 5 (2010) 617. [3] M. Pollini, et al., European Patent No. EP1986499 (2008).

Resume : Introduction The uncontrollable growth of tumor cells results in decreased levels of oxygen and increased levels of reactive oxygen species (ROS) in the tumor cell microenvironment. Hence, systems that are able to scavenge ROS while increasing oxygen levels represent a promising approach to treat cancer. In the present work, poly(lactide-co-glycolide) (PLGA) microspheres coated with collagen and functionalized with manganese dioxide (MnO2) nanoparticles (NPs) were developed. On the one hand, MnO2 NPs will scavenge H2O2 and generate O2 while on the other hand, the external collagen layer will protect the microspheres from a variety of biological enemies (i.e. enzymes) and will eliminate the toxicity of the embedded NPs. Materials & Methods MnO2 NPs and PLGA/Collagen hollow microspheres were successfully synthesized and characterized using XRD, FT-IR, SEM, TEM and DLS. An oxygen sensor was used to measure the increase in oxygen levels while an H2O2 assay kit was used to assess the scavenging effect of the whole system. Cytotoxicity of the PLGA/collagen hollow microspheres functionalized with NPs was carried out on fibroblast, MCF-7 and Hela cells. Results & Discussion PLGA was successfully activated using carbodiimide chemistry. The microspheres were fabricated using an oil-in-water emulsion technique and subsequently coated with collagen. MnO2 NPs were added to the coating solution resulting in their embedment inside the collagen layer. Oxygen was generated upon the reaction of the NPs in the outer layer with H2O2. PLGA/collagen microspheres with MnO2 NPs were found to scavenge H2O2 in a concentration dependent manner. In vitro studies on fibroblast, MCF-7 and Hela cells after treatment with H2O2 before and after treatment with MnO2 NPs demonstrated that metabolic activity was higher after reaction with MnO2 but no toxicity was observed. Conclusions The microspheres developed in this work were able to efficiently scavenge ROS and generate oxygen in a concentration dependent manner. In vitro studies indicated that the developed microspheres were able to protect cells from the toxic effect of hydrogen peroxide.

Resume : INTRODUCTION Bioceramic scaffolds are typically characterised by exceptional compressive strength (Young?s modulus) and very low elasticity. From a bone perspective they exhibit excellent biocompatibility due to their chemical and structural similarity to the mineral phase of native bone1. The main deficiency in these materials is their brittle nature, which poses a concern in high load-bearing biological applications2. Polymers on the other hand have insufficient strength to meet the mechanical requirements of bone graft substitutes in vivo, whilst suffering from the inability to induce mineralisation3. However through advances in polymer synthesis technologies, particularly with regards to controlled free radical polymerisation and scaffolding techniques, polymers can be developed by tailoring their physicochemical properties4 and as such they do not suffer from brittleness. Hence the current study will assess a combination of polymers and bioceramics in a composite form to determine if this combination acts synergistically to overcome the limitations of either individual component. MATERIALS AND METHODS Composites of polyethylene glycol dimethacrylate (PEGMDA) and acrylic acid (AA) pre-polymerised solution were added to a ceramic scaffold composing of ?-wollastonite and ?-tricalcium phosphate (W-TCP). The pre-polymerised solution containing the ceramic scaffold was fabricated using free radical photopolymerisation technique. The resultant composite material was characterised by FTIR, SEM, swelling studies, gel fraction and compression testing. Statistical analysis of the data was performed using a Tukey test Post hoc test. RESULTS FTIR and SEM analysis illustrated that the polymer had penetrated the ceramic scaffold. Swelling studies in buffer solution pH 7.4 showed a decrease swelling when the polymer was incorporated into the ceramic scaffold. Additionally incorporation of the pre-polymerised solution into the ceramic scaffold was found not to disrupt the network connectivity and in all cases gel formation occurred. Compression testing demonstrated that the incorporation of the polymer into the ceramic scaffold resulted in an increase in the mechanical properties. DISCUSSION AND CONCLUSION Cortical bone has a compressive strength of 100-200MPa5, hence providing scaffolds with the required mechanical stability for use as a bone substitute material is essential. Compression results revealed a significant increase in the compressive strength of the polymer-ceramic composite when compared to the polymer control hydrogel (p< 0.05). This improvement in mechanical properties may be attributed to the reduction in the degree of swelling, the ceramic scaffold absorbing the compressive load and the polymer within the scaffold distributing the load hence reducing brittleness of the ceramic. REFERENCES 1. O'Brien, Biomaterials and scaffolds for tissue engineering. Materials Today 14(3) 1-21, 2011 2. Shrivats (et al.), Bone tissue engineering: state of the union. Drug Discovery Today 19(6) 781-786, 2014 3. El-Sherbiny and Yacoub, Hydrogel scaffolds for tissue engineering: Progress and challenges. Global Cardiology Science and Practice 38 316-342, 2013 4. Vo (et al.), Strategies for controlled delivery of growth factors and cells for bone regeneration. Advanced Drug Delivery Review 64 1292-1309, 2012 5. Bose (et al.), Recent advances in bone tissue engineering scaffolds. Trends in Biotechnology 30 (10), 546-554 2012 Acknowledgments This work has been supported through COST action NEWGEN STSM funding and AIT Presidents Seed Fund. Authors want to acknowledge the Ministry of Economy and Competitiveness of Spain for financial support under projects MAT2013-48426-C2-1R and CSIC-201460E066.

Resume : Tendon injuries are a leading cause of disability in athletes, active working people and elder population worldwide. Partial or total loss of tendon functionality is mainly caused by a poor alignment of collagen fibrils in scar tissue, resulting in significant mechanical limitation of repaired tendons. Since tendon morphology and functionality are intrinsically associated, tendon engineered scaffolds should mimic the anisotropic structural architecture of native tendons to support a complete regeneration of damaged tissues. Thus, sophisticated scaffolds with aligned structural features were fabricated using micro-fabrication technologies. The microstructures of starch and polycaprolactione (SPCL) matrices created by 3D printing technologies allow to control the spatial distribution of cells and guide cell behavior towards a tenogenic phenotype. Moreover, the integration of magnetic nanoparticles (MNPs) in anisotropic scaffolds to be remotely controlled by an external magnetic field can further contribute for 3D systems with improved functionality and added value for tendon regeneration. Stem cells from adipose tissue laden in smart magnetic scaffolds naturally respond to magnetic forces synthesizing a Tenascin and Collagen I rich matrix. The developed magnetic scaffolds were biocompatible and showed good in vivo integration with surrounding tissues. Considering that matrix stiffness is also a major stimulus driving cell fate and organization, glycosaminoglycan fiber hydrogels produced by combining microfluidics with polyelectrolyte interactions may be an interesting approach towards the regeneration of tendons. Fiber hydrogels replicate topographical stimulation and elastic properties of tendon, in which tendon derived cells were found to be homogeneously distributed along the fibers, being able to produce the matrix components Collagen I and Tenascin. Altogether, the developed strategies suggest that magnetic stimulation and biomimetic scaffolds with topographic cues may trigger the next generation of implantable devices for tendon regenerative therapies. The authors wish to acknowledge the financial support of the Recognize project (UTAP-ICDT/CTM-BIO/0023/2014)

Resume : As a category of new and promising regulation approaches for stem cell fates, physical cues, including electric or magnetic field, photo irradiation, acoustics, and even pressure and strain, have been attracted increasing attention most recently. With progress of research in this field, some clues connecting the physical signal and bio pathway for stem cell differentiation have been discovered. However, more phenomena are still waiting for discovered. Compared with bio growth factor, or chemical signal, physical signal is very easy to be controlled, not only for the quantity, but also can be localized in a specific area. Therefore, physical techniques for tuning stem cell differentiation is more benefit for stem cell stem-cell therapy and tissue engineering. However, the great obstacle for study and application of physical signal tuned stem cell differentiation is how to apply physical signal on stem cells. In these cases, design and preparation of nanostructure by full use of the physical properties of the materials become the most important to provide a tools to mediate the interaction between physical signal and stem cells. Recent year, our group paid much attention to regulation of stem cell fate by physical cues, including the electric pulse simulation, surface charge of the materials, ultrasonic transferred electrons, magnetic transferred alternated electric field, and light with different wavelength. To realize these investigation, we designed and synthesized different nanostructures and nanoparticles specialized for different studies. These materials act as media, which connects the stem cell and physical signals. In this talk, we will report the above works, introduce how to design and synthesis nanostructure or nanoparticle system to connect the primary physical signal to stem cell, and how the physical signal tune the fate of the stem cells. We believe that the regulation effect of physical signal will attract more attention, and will have great impact for design and application of biomaterials, especially for tissue engineering scaffold, and will bring great progress in tissue regeneration medicine.

Resume : The driven hypothesis of scaffold-free, cell-based therapies is that replacement, repair and restoration of tissue function can be accomplished best by recruiting cells’ inherent proficiency to create their own tissue-specific extracellular matrix with precision and stoichiometric efficiency still unmatched by man-made devices. Such therapeutic strategies require removal of cells from their optimal, in regards to topography, stiffness, packing density, oxygen tension and mechanical loading tissue context, and propagation thereof in far from physiological in vitro environment. However, bereft of their tissue context, cells perform poorly, lose their functionality, and with it, their therapeutic potential. To this end, research efforts have been directed towards reconstruction of more functional in vitro microenvironments by modulating substrate topography; substrate stiffness; macromolecular crowding; oxygen tension; and mechanical loading in order to provide an optimal in vitro niche that will closely mimic cells’ natural in vivo conditions and thus facilitate maintenance of their therapeutic potential. Herein, the influence of topographical features, macromolecular crowding and oxygen tension will be discussed as means to control cellular function and consequently enable clinical translation and commercialization of cell-based therapies.

Resume : The morphology and dimensions of bioactive materials are essential attributes to promote tissue culture. Nanofibrous structures have excellent potential to be used as scaffolds. On the other hand, bioactive glasses with applications in regenerative medicine may present antibacterial properties, which depend on glass composition, concentration and the microorganisms tested. Likewise, their morphology may influence their antibacterial activity too. In the present work, the laser spinning technique was used to produce bioactive glass nanofibers of two different compositions: 45S5 Bioglass® and ICIE16M, bioactive glass doped with Zn and Sr. Biocompatibility was analyzed by means of cell viability tests performed with BALB/3T3 cell line. Their antibacterial activity against S. aureus was tested by culturing them in dynamic conditions. Bacterial growth index profiles during the first days of experiment can be explained by the variations in the pH values of the media. The bactericidal effect of the doped nanofibers at longer times is justified by the release of Zn and Sr ions.

Resume : Enhanced characteristics of implants relies on the ability to tune and control the surface of materials. Although Titanium and its alloy proved to be reliable materials for implants, the need of improved bio-interfaces emerged in the last decades in the field of orthopedic implants. In this work, Matrix Assisted Pulsed Laser Evaporation (MAPLE) method was used for obtaining thin complex hybrid coatings of lactoferrin (Lf) and hydroxyapatite (HA) on Ti based alloy. Scanning Electron Microscopy, Atomic Force Microscopy were used for morphologycal films characterization, while the functional groups in the MAPLE-deposited coatings were verified by FTIR measurements. THP-1 cells stimulated with bacterial endotoxins constituted the in vitro inflammation model for analysing the potential cellular inflammatory response induced by biomimetic coated bioalloy. THP-1 cells adhered preferentially to te hybrid Lactoferrin -hydroxyapatite coated surfaces when compared to films embedded with HA or Lf alone. In the presence of LPS, a decrease in the total number of cells was observed irrespective of surface covering. Hybrid biomimetic Lf-HA functionalized bioalloy proved to be active in modulation of immune response, thus being a good candidate to improve the performance of bone implants. Acknowledgments: The research leading to these results has received funding from the Romanian Ministry of National Education, CNCS ? UEFISCDI, under the projects PN-II-PT-PCCA-2013-4-199, PN-II-RU-TE-2014-4-2434 and PN09-39

Resume : The slow crack growth behavior of yttria-stabilized tetragonal ziconia (Y-TZP) dental ceramic was investigated in several environments and crack velocity curves (V?KI curves) were obtained under static fatigue using Vickers indentation method. The aim of this study was to evaluate the environmentally slow crack growth mechanisms of Y-TZP dental ceramic. Additionally, Confocal Raman spectroscopy has been used for the quantitative assessments of phase transformation during slow crack growth in Y-TZP. Environmental metastability of Y-TZP take place a tetragonal- to-monoclinic (t ? m) phase transformation and proceed with time. We reported for the first time the relative amount of surface phase-transformation fractions at different stages of slow crack growth. Transformation at the surface of ground specimens leads to surface compressive induced stresses and eventually affect the fatigue behavior. The Raman spectroscopic assessment may provide a new angle of view in understanding the fatigue behavior of zirconia ceramics in different environments as well as in developing new zirconia-based biomaterials characterized by superior properties.

Resume : Wearable biosensors attachable on the skin have been developed for real time healthcare. Since the more biological information (e.g., physiological ions, disease markers, etc) are beneath the skin in the intravascular, only a few of physiological information can be detected on the skin by using such sensors. The more information can be obtained if the sensors can be worked under the skin. Herein, we reported a patterned microprojection skin patch (PMSP) which can project the skin and detect intravascular cholera toxin without incision. Si micropillar patterns with height of 800 ?m were prepared by DRIE (Deep Reactive-Ion Etching) and wet etch process. CMOS (Complementary Metal-Oxide Semiconductor) process was then used to form an electrical circuit that makes each pillar works as individual electrode. The micropillar electrodes were coated with peptides that work as receptor for the cholera toxin. The electrodes were then connected to FPCB (flexible printed circuit board) and impedance analyzer. The sensing test was carried out by projection of the micropillar electrodes into the uninfected explanted human dermal tissue, and then injection of diluted cholera toxin to the tissue by syringe pump. The patch showed change of impedance and/or capacitance between the micropillar electrodes by the reaction of peptides with intravascular cholera toxin. The signal to noise ratio is high and stable enough for PMSP working as biosensors that can work under the skin.

Resume : Introduction Collagen is the main component of ECM with 29 types identified. Type II collagen presents supportive function in cartilaginous tissue. Since type II collagen is the major component of articular cartilage in the knee joint, the potential of an implantable type II collagen hydrogel as a cell carrier for cartilage regeneration is being investigated in this project. In this work we isolated and characterized type II collagen from porcine cartilage and cartilaginous fish through pepsin digestion. Pepsin has been discovered to cleave the telopeptide region of the collagen molecule, pepsin treated collagen extraction ensures higher collagen yield and telopeptide-free collagen reveals biocompatible, biodegradable and less toxic function. Type II collagen was crosslinked with 4arm and 8arm StarPEG. We hypothesize that pure type II collagen can be obtained from cartilaginous tissue and 8arm StarPEG crosslinking will increase the mechanical, biological and biomedical properties of collagen hydrogel. Materials and methods Type II collagen was extracted from porcine trachea, ear, articular cartilage and cartilaginous fish through acid-pepsin digestion at 4ºC. The structure of collagen sponge was shown by scanning electron microscopy (SEM). SDS-PAGE was used to reveal the number and size of collagen chains. Thermal stability was tested by differential scanning calorimetry (DSC). Enzymatic degradation was assessed by collagenase assay. TNBSA assay will be carried out to test the free amine group in crosslinked collagen hydrogel. Conclusions: Pure type II collagen can be extracted from porcine cartilage and cartilaginous fish. Further biological assays will be carried out to characterize collagen enzymatic degradation and thermal stability. Reference: 1.Collin, E. C., et al. Biomaterials 32.11 (2011): 2862-2870 Acknowledgments: The authors would like to thank Teagasc, Irish Agriculture and Food Development Authority for providing financial support for this project.

Resume : Silk has been a highly prized material for medicinal and skincare remedies since ancient times. It is a natural and versatile material that can be used for a wide range of applications. In recent years, more efforts have been directed towards modifying properties or enhancing functionalities of silk by integrating with various materials, and the resulting functional silk has been used as a key component in diagnostic and therapeutic devices where biocompatibility, degradability and flexibility are required. We have developed a green method to produce a new class of functional silk and demonstrated its utility as biocompatible tissue engineering scaffolds, color cosmetics and skincare ingredients. Moreover, we were able to enhance the mechanical properties of silk, rendering it potentially useful for biomedical tools such as surgical sutures.

Resume : In the present study, a facile synthesis route was developed to prepare surface functioanlized superparamagnetic Cobalt zinc ferrite (CZF) magnetic nanoparticles (MNPs) by using triethylene glycol (TEG) as reducing agent and surface modifier ligand. Initially structural, morphological, and magnetic characterization were carried out in order to confirm their size, poly dispersity, colloidal stability, and magnetic property. Fourier transform infrared spectroscopy (FTIR) confirmed the presence of triethylene glycol (TEG) on the surface of CZF MNPs. The CZF MNPs are of superparamagnetic in nature with high saturation magnetization, good colloidal stability, high specific absorption rate (SAR), and excellent biocompatibility. All these properties are crucial, for their use as nanomedicine in cancer theranostics such as magnetic fluid hyperthermia (MFH) treatment; which is considered as one of the most promising cancer therapy. The prepared CZF MNPs are found biocompatible with MCF7 (human breast cancer) and L929 (mouse fibroblast) cell lines and Vero cell line (monkey kidney cell line), when tested by MTT and SRB assays. Cell particle interaction was studied in depth, by using multiple staining techniques combined with confocal microscopy. Finally, an In vitro hyperthermia experiment was carried on MCF7 cells, resulting in death of MCF7 cells up to 80% within 60 min. The research demonstrates that, the prepared CZF MNPs can be used as a potential candidate for effective MFH treatment for cancer cell death. Reference: 1. R.A. Bohara, N.D. Thorat, A.K. Chaurasia, S.H. Pawar, RSC Adv., 5, 47225 (2015) Corresponding Author: Raghvendra A Bohara Centre for Interdisciplinary Research, D.Y. Patil University, Kolhapur Email : raghvendrabohara@gmail.com

Resume : Designing bulk polymer composite materials with firmly embedded nanofillers having good biocompatibility,high bactericidal activity and large scale production capability is considered of technological importance.Biodegradable polylactic acid (PLA) with 18 wt% hydroxyapatite nanorod (nHA) and silver nanoparticle (AgNP) of different loadings were fabricated by melt-compounding process. Hybridizing nHA with AgNP fillers in the PLA matrix permitted efficient attachment and proliferation of osteoblasts and good bactericidal ability of the resulting nanocomposites. This study aimed to evaluate the biodegradation, antibacterial ability, bioactivity and cytotoxicity of melt-compounded PLA/18% nHA?Ag hybrids using solution immersion, water contact angle, agar disk diffusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and biomineralization measurements. Weight-loss and water contact angle measurements showed that the nHA and Ag nanofillers increase the degradation rate and hydrophilicity of PLA, respectively; AgNPs were more effective than nHA for those tests. Disk diffusion test results demonstrated that the PLA/18% nHA?Ag hybrids show high bactericidal activity against Escherichia coli and moderate activity against Staphylococcus aureus. MTT test results revealed that high AgNP contents (18 and 25 wt%) in the PLA hybrids inhibit the proliferation of osteoblasts. However, composite hybrids with low loading Ag levels (2 and 6 wt%) showed good biocompatibility. Such hybrids maintained a good balance between antibacterial activity and cytocompatibility. Biomineralization test revealed that a dense apatite layer can be fully developed on the surfaces of PLA/18% nHA?Ag hybrids. The development of industrially scalable, efficient and cost effective polymer composite hybrids with good osteoconductivity and great bactericidal activity opens new perspective for bone tissue engineering applications.

Resume : The integrity and function of the periodontium can be compromised by traumatic injuries or periodontitis. Currently available clinical therapies are able to stop the progression of periodontitis and allow the healing of periodontal tissue. However an optimal strategy capable of restoring the anatomy and functionality of the lost periodontal tissue is still to be achieved. Herein is proposed the development of an injectable hydrogel system able to release a growth factors and cells to the periodontal defect. This injectable system is based on a photocrosslinkable hydrogel, prepared from methacrylated Hyaluronic Acid (me-HA) and incorporating Platelet Lysate (PL). The delivery of growth factors and cells in situ is expected to enhance regeneration of the periodontium. Various formulations of me-HA containing increasing PL concentrations were studied for achieving the formation of stable photocrosslinkable hydrogels. The produced hydrogels were subsequently characterized to assess mechanical properties, degradation, protein/growth factor release profile, antimicrobial activity and response towards human Periodontal Ligament fibroblasts (hPDLFs). The results demonstrated that it was possible to obtain stable photocrosslinkable hydrogels incorporating different amounts of PL that can be released in a sustained manner. Furthermore, the incorporation of PL improved the viscoelastic properties of the hydrogels and enhanced their resilience to the degradation by hyaluronidase (HAase). Additionally, the PL showed to provide antimicrobial properties. Finally, hPDLFs, either seeded or encapsulated into the developed hydrogels, showed enhanced proliferation over time, proportionally to the increasing amounts of PL present in the hydrogel formulations.

Resume : Due to an aging population and increased of sports-related injuries, musculoskeletal disorders have become one of the major health concerns in the Western Countries. Current treatments, typically rely on donor tissues and do not provide optimum therapy. This has prompted orthopedic implants as the most reliable solution, thus leading to an increasing behavior of implant patients over the past years. Implant-associated infections are a major clinical problem and one of the main causes of implant failure. Although antibiotics can be administered systemically in clinical therapy, a more controlled release needs to be achieved in order to conclude to the optimum implant efficacy. In this work, a novel Drug Delivery Nanoplatform of Polycaprolactone scaffolds loaded with vancomycin drug, has been fabricated via Electrospinning process. Titanium implants? surface have been modified with fibrous coatings and the surface structure of the implants was observed using Scanning Electron Microscope. The efficacy and cytotoxicity of the titanium implants were investigated in vitro, using MTT proliferation assay and Methylene Blue staining, thus showed excellent cytocompatibility behavior. The release behavior of vancomycin from nanofiber structures exhibited a biphasic release pattern, with an initial burst on the 1st day, followed by a slow and controlled release 30 days period long, where was released above 85% of vancomycin. Due to the unique advantages of the polymeric fiber-based structures to control drug release kinetics and preserve a sustained release onto the implant?s surface, vancomycin-coated titanium implants may be a promising approach to prevent and treat implant-associated infections.