Bioactive coating on titanium implants modified by Nd:YVO4 laser

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Revêtement anti-apoptotique à base de chondroïtine sulfate : vers un stent-graft bioactif

La réparation endovasculaire (EVAR) est une technique minimalement invasive permettant de traiter l’anévrisme de l’aorte abdominale (AAA) par l’entremise d’un stent- graft (SG). L’utilisation d’EVAR est actuellement limitée par de fréquentes complications liées à une guérison inadéquate autour de l’implant. Ce manque de guérison est principalement dû au type de recouvrement polymérique des SG, au milieu pro-apoptotique des AAA et à l’accès réduit aux nutriments et à l’oxygène après EVAR. L’objectif de cette thèse consistait à concevoir un revêtement bioactif permettant d’inhiber l’apoptose et stimuler la croissance des cellules musculaires lisses vasculaires (CMLV), pour ainsi favoriser la guérison des tissus vasculaires autour des SG. La chondroïtine-4-sulfate (CS) a d’abord été choisie, car elle a été identifiée comme un médiateur important de la réparation vasculaire. Il a été démontré que la CS en solution influence directement la résistance à l’apoptose des CMLV, en plus de favoriser la différenciation myofibroblastique chez les fibroblastes. Dans le cadre de ce projet, un premier revêtement à base de CS et de collagène a été créé. Bien que le revêtement permettait d’induire une résistance à l’apoptose chez les CMLV, il se désintégrait trop rapidement dans des conditions aqueuses. Une nouvelle méthodologie a donc été adaptée afin de greffer la CS directement sur des surfaces aminées, à l’aide d’un système utilisant un carbodiimide. Dans le but d’accroître la croissance des CMLV à la surface des revêtements, le facteur de croissance de l’épiderme (EGF) a ensuite été sélectionné. En plus de ses propriétés mitogéniques et chimiotactiques, l’EGF stimule la production d’éléments de la matrice extracellulaire, comme le collagène et la fibronectine. De plus, l’activation du récepteur de l’EGF inhibe également l’apoptose des CMLV. L’EGF a donc été greffé sur la CS. Le revêtement de CS+EGF a démontré une bonne uniformité et bioactivité sur des surfaces de verre aminé. iii iv Dans une 3ème étape, afin de permettre de transposer ce revêtement bioactif sur des implants, plusieurs méthodes permettant de créer des groupements d’amines primaires sur les biomatériaux polymériques comme le PET ou le ePTFE ont été étudiées. La polymérisation par plasma a été choisie pour créer le revêtement CS+EGF à la surface de PET. Une fois de plus, celui-ci a permis d’inhiber l’apoptose des CMLV, dans des conditions pro-apoptotiques, et de favoriser la croissance des cellules. Le revêtement de CS et d’EGF, déposé sur des surfaces aminées, possède des caractéristiques biologiques intéressantes et semble donc prometteur pour favoriser une meilleure guérison autour des SG.

Development of nanohydrogels as a vehicle for the release of bioactive compounds in food matrices

PhD in Chemical and Biological Engineering

Bioactive films produced from self-assembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions

The development of versatile bioactive surfaces able to emulate in vivo conditions is of enormous importance to the future of cell and tissue therapy. Tuning cell behaviour on two-dimensional surfaces so that the cells perform as if they were in a natural three-dimensional tissue represents a significant challenge, but one that must be met if the early promise of cell and tissue therapy is to be fully realised. Due to the inherent complexities involved in the manufacture of biomimetic three-dimensional substrates, the scaling up of engineered tissue-based therapies may be simpler if based upon proven two-dimensional culture systems. In this work, we developed new coating materials composed of the self-assembling peptide amphiphiles (PAs) C16G3RGD (RGD) and C16G3RGDS (RGDS) shown to control cell adhesion and tissue architecture while avoiding the use of serum. When mixed with the C16ETTES diluent PA at 13 : 87 (mol mol-1) ratio at 1.25 times 10-3 M, the bioactive {PAs} were shown to support optimal adhesion, maximal proliferation, and prolonged viability of human corneal stromal fibroblasts ({hCSFs)}, while improving the cell phenotype. These {PAs} also provided stable adhesive coatings on highly-hydrophobic surfaces composed of striated polytetrafluoroethylene ({PTFE)}, significantly enhancing proliferation of aligned cells and increasing the complexity of the produced tissue. The thickness and structure of this highly-organised tissue were similar to those observed in vivo, comprising aligned newly-deposited extracellular matrix. As such, the developed coatings can constitute a versatile biomaterial for applications in cell biology, tissue engineering, and regenerative medicine requiring serum-free conditions.

Self-assembly of a dual functional bioactive peptide amphiphile incorporating both matrix metalloprotease substrate and cell adhesion motifs

We describe a bioactive lipopeptide that combines the capacity to promote the adhesion and subsequent self-detachment of live cells, using template-cell-environment feedback interactions. This self-assembling peptide amphiphile comprises a diene-containing hexadecyl lipid chain (C16e) linked to a matrix metalloprotease-cleavable sequence, Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, and contiguous with a cell-attachment and signalling motif, Arg-Gly-Asp-Ser. Biophysical characterisation revealed that the PA self-assembles into 3 nm diameter spherical micelles above a critical aggregation concentration (cac). In addition, when used in solution at 5–150 nM (well below the cac), the PA is capable of forming film coatings that provide a stable surface for human corneal fibroblasts to attach and grow. Furthermore, these coatings were demonstrated to be sensitive to metalloproteases expressed endogenously by the attached cells, and consequently to elicit the controlled detachment of cells without compromising their viability. As such, this material constitutes a novel class of multi-functional coating for both fundamental and clinical applications in tissue engineering.

Biological Performance of Chemical Hydroxyapatite Coating Associated With Implant Surface Modification by Laser Beam: Biomechanical Study in Rabbit Tibias

Purpose: Considering the potential of the association between laser ablation and smaller scale hydroxyapatite (HA) coatings to create a stable and bioactive surface on titanium dental implants, the aim of the present study was to determine, by the removal torque test, the effects of a surface treatment created by laser-ablation (Nd:YAG) and, later, thin deposition of HA particles by a chemical process, compared to implants with only laser-ablation and implants with machined surfaces.Materials and Methods: Forty-eight rabbits received I implant by tibia of the following surfaces: machined surface (MS), laser-modified surface (LMS), and biomimetic hydroxiapatite coated surface (HA). After 4, 8, and 12 weeks of healing, the removal torque was measured by a torque gauge. The surfaces studied were analyzed according to their topography, chemical composition, and roughness.Results: Average removal torque in each period was 23.28, 24.0, and 33.85 Ncm to MS, 33.0, 39.87, and 54.57 Ncm to LMS, and 55.42, 63.71 and 64.0 Ncm to HA. The difference was statistically significant (P < .05) between the LMS-MS and HA-MS surfaces in all periods of evaluation, and between LMS-HA to 4 and 8 weeks of healing. The surface characterization showed a deep, rough, and regular topography provided by the laser conditioning, that was followed by the HA coating.Conclusions: Based on these results, it was possible to conclude that the implants with laser surface modification associated with HA biomimetic coating can shorten the implant healing period by the increase of bone implant interaction during the first 2 months after implant placement. (C) 2009 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 67:1706-1715, 2009

Growth of calcium phosphate coating on Ti-7.5Mo alloy after anodic oxidation

Titanium and its alloys are widely used as biomaterials due to their mechanical, chemical and biological properties. To enhance the biocompatibility of titanium alloys, various surface treatments have been proposed. In particular, the formation of titanium oxide nanotubes layers has been extensively examined. Among the various materials for implants, calcium phosphates and hydroxyapatite are widely used clinically. In this work, titanium nanotubes were fabricated on the surface of Ti-7.5Mo alloy by anodization. The samples were anodized for 20 V in an electrolyte containing glycerol in combination with ammonium fluoride (NH4F, 0.25%), and the anodization time was 24 h. After being anodized, specimens were heat treated at 450 °C and 600°C for 1 h to crystallize the amorphous TiO2 nanotubes and then treated with NaOH solution to make them bioactive, to induce growth of calcium phosphate in a simulated body fluid. Surface morphology and coating chemistry were obtained respectively using, field-emission scanning electron microscopy (FEG-SEM), AFM and X-ray diffraction (XRD). It was shown that the presence of titanium nanotubes induces the growth of a sodium titanate nanolayer. During the subsequent invitro immersion in a simulated body fluid, the sodium titanate nanolayer induced the nucleation and growth of nano-dimensioned calcium phosphate. It was possible to observe the formation of TiO2 nanotubes on the surface of Ti-7.5Mo. Calcium phosphate coating was greater in the samples with larger nanotube diameter. These findings represent a simple surface treatment for Ti-7.5Mo alloy that has high potential for biomedical applications. © (2013) Trans Tech Publications, Switzerland.

Influence of Edible Coating on the Drying and Quality of Papaya (Carica papaya)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Poly(epsilon-caprolactone) nanocapsules carrying the herbicide atrazine: effect of chitosan-coating agent on physico-chemical stability and herbicide release profile

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bioactive compounds and physical and chemical characteristics of mini tomatoes: influence of postharvest treatments

Tomatoes are among the most cultivated and used vegetables in the world. They are very succeptible to post harvest losses due to high perishability, therefore the use of postharvest treatments may contribute to conservation of this fruit, however the treatments might affect significantly physico-chemical, sensory and nutritional characteristics of tomatoes. Given the perishability of tomato and the economic importance of small tomato fruits, the purpose of the present study was to determine the effect of gamma radiation, carnauba coating and 1-MCP treatments on tomato fruit quality during storage. The study may be divided into two parts. In the first, mini tomatoes cv. Sweet Grape were harvested at breaker stage, divided into 4 grous and treated with gamma radiation (0.6 kGy), carnauba coating (1 L 1000 kg-1) and 1-MCP (500 nL L-1) and then stored at 25±2°C for 30 days with a control group of tomatoes. In the seconnd part, tomatoes harvested at light-red stage were submitted to the same treatments and storage period. Every 6 days tomatoes were evaluated for color modifications, fruit firmness, souble and total pectin (only for light-red tomatoes), mass loss, titratable acidity (TA), soluble solids (SS), SS/TA ratio, carotenoids profile, formation of lycopene isomers, total phenolic compounds, ascorbic acid and antioxidant capacity. For tomatoes harvested at breaker stage and submitted to the treatments the results showed mass loss was delaying mainly by carnauba wax, and to a lesser extend by 1-MCP. Fruit firmness were better retained for 1-MCP treated fruits and carnauba treatment showed a transient effect in preserving fruit firmness. SS/TA of tomatoes treated with gamma radiation and carnauba presented no differences from control values, and were lower with the application of 1-MCP. Color was negatively affected by 1-MCP and earlier changed (6th day) when gamma radiation was applied. In relation to bioactive compounds of tomatoes harvest at breaker stage, results indicated gamma radiation and 1-MCP decreased the final content of lycopene and produced more (Z)-isomers of lycopene. Gamma radiation also induced a decreased in ?-carotene and an increased in phenolic compounds by the end of storage period. 1-MCP treatment promoted a slow down increase in ascorbic acid content during storage. Antioxidant capacity of the hydrophilic fraction was not dramatically affected by treatments and the lipophilic fraction was lower, especially for 1-MCP fruits. In addition, contents of ?-carotene, lycopene, (Z)-isomers of lycopene, ascorbic acid and antioxidant capacity increased during the period of storage while contents of lutein and phenolic compounds tended to decrease. Regarding tomatoes harvest at light-red stage, the most effective treatments for delaying fruit firmness and mass loss was carnauba and 1-MCP, while gamma radiation was the treatment with higher mass loss and the less fruit firmness, which could be associated with the higher solubilization of pectins promoted by radiation treatment. Color (L* and Hue) was mainly affected by 1-MCP treatment which delayed color development, however, by the end of storage, the values were not different from the other treatments. SS/TA ratio was lower for fruits treated with 1-MCP and TA was not so dramatically affected by treatments. Furthermore, mini tomatoes harvested at light-red stage, demonstrated irradiation induced changes in the final content of lycopene, increasing it, and formed less (13Z)-lycopene, while 1-MCP and carnauba coating slow down the increase in lycopene and slown down the decrease of ascorbic acid and phenolic compounds. Antioxidant capacity of lipophilic fraction was not affected by treatments and the hydrophilic fraction was lower for irradiated fruits only on day 0 as well as phenolic compounds. In the other days, no differences among treatments were observed for hydrophilic antioxidant capacity. Considering the results, the best combination of SS and TA and fruit preservation for mini tomatoes harvest at breaker stage was promoted by carnauba coating, which seems to be the treatment that causes fewer changes in bioactive compounds of breaker tomatoes. However, when mini tomatoes were harvested at light-red stage, SS/TA ratio and color were better and, to preserve the quality of these fruits, besides carnauba coating, 1-MCP also could be indicated

Multifunctional bioactive glass scaffolds coated with layers of poly(d,l-lactide-co-glycolide) and poly(n-isopropylacrylamide-co-acrylic acid) microgels loaded with vancomycin

A new family of multifunctional scaffolds, incorporating selected biopolymer coatings on basic Bioglass® derived foams has been developed. The polymer coatings were investigated as carrier of vancomycin which is a suitable drug to impart antibiotic function to the scaffolds. It has been proved that coating with PLGA (poly(lactic-co-glycolic acid)) with dispersed vancomycin-loaded microgels provides a rapid delivery of drug to give antibacterial effects at the wound site and a further sustained release to aid mid to long-term healing. Furthermore, the microgels also improved the bioactivity of the scaffolds by acting as nucleation sites for the formation of HA crystals in simulated body fluid. © 2013 Elsevier B.V. All rights reserved.

Novel bioactive scaffolds incorporating nanogels as potential drug eluting devices

Big advances are being achieved in the design of new implantable devices with enhanced properties. For example, synthetic porous three-dimensional structures can mimic the architecture of the tissues, and serve as templates for cell seeding. In addition, polymeric nanoparticles are able to provide a programmable and sustained local delivery of different types of biomolecules. In this study novel alternative scaffolds with controlled bioactive properties and architectures are presented. Two complementary approaches are described. Firstly, scaffolds with nanogels as active controlled release devices incorporated inside the three-dimensional structure are obtained using the thermally induced phase separation (TIPS) method. Secondly, a novel coating method using the spraying technique to load these nanometric crosslinked hydrogels on the surface of two-dimensional (2D) and three-dimensional (3D) biodegradable scaffolds is described. The scanning electron microscopy (SEM) images show the distribution of the nanogels on the surface of different substrates and also inside the porous structure of poly-a-hydroxy ester derivative foams. Both of them are compared in terms of manufacturability, dispersion and other processing variables.

Intensification Of Bioactive Compounds Extraction From Medicinal Plants Using Ultrasonic Irradiation.

Extraction processes are largely used in many chemical, biotechnological and pharmaceutical industries for recovery of bioactive compounds from medicinal plants. To replace the conventional extraction techniques, new techniques as high-pressure extraction processes that use environment friendly solvents have been developed. However, these techniques, sometimes, are associated with low extraction rate. The ultrasound can be effectively used to improve the extraction rate by the increasing the mass transfer and possible rupture of cell wall due the formation of microcavities leading to higher product yields with reduced processing time and solvent consumption. This review presents a brief survey about the mechanism and aspects that affecting the ultrasound assisted extraction focusing on the use of ultrasound irradiation for high-pressure extraction processes intensification.

Paired Evaluation Of Calvarial Reconstruction With Prototyped Titanium Implants With And Without Ceramic Coating.

To investigate the osseointegration properties of prototyped implants with tridimensionally interconnected pores made of the Ti6Al4V alloy and the influence of a thin calcium phosphate coating. Bilateral critical size calvarial defects were created in thirty Wistar rats and filled with coated and uncoated implants in a randomized fashion. The animals were kept for 15, 45 and 90 days. Implant mechanical integration was evaluated with a push-out test. Bone-implant interface was analyzed using scanning electron microscopy. The maximum force to produce initial displacement of the implants increased during the study period, reaching values around 100N for both types of implants. Intimate contact between bone and implant was present, with progressive bone growth into the pores. No significant differences were seen between coated and uncoated implants. Adequate osseointegration can be achieved in calvarial reconstructions using prototyped Ti6Al4V Implants with the described characteristics of surface and porosity.

Combining Xanthan And Chitosan Membranes To Multipotent Mesenchymal Stromal Cells As Bioactive Dressings For Dermo-epidermal Wounds.

The association between tridimensional scaffolds to cells of interest has provided excellent perspectives for obtaining viable complex tissues in vitro, such as skin, resulting in impressive advances in the field of tissue engineering applied to regenerative therapies. The use of multipotent mesenchymal stromal cells in the treatment of dermo-epidermal wounds is particularly promising due to several relevant properties of these cells, such as high capacity of proliferation in culture, potential of differentiation in multiple skin cell types, important paracrine and immunomodulatory effects, among others. Membranes of chitosan complexed with xanthan may be potentially useful as scaffolds for multipotent mesenchymal stromal cells, given that they present suitable physico-chemical characteristics and have adequate tridimensional structure for the adhesion, growth, and maintenance of cell function. Therefore, the purpose of this work was to assess the applicability of bioactive dressings associating dense and porous chitosan-xanthan membranes to multipotent mesenchymal stromal cells for the treatment of skin wounds. The membranes showed to be non-mutagenic and allowed efficient adhesion and proliferation of the mesenchymal stromal cells in vitro. In vivo assays performed with mesenchymal stromal cells grown on the surface of the dense membranes showed acceleration of wound healing in Wistar rats, thus indicating that the use of this cell-scaffold association for tissue engineering purposes is feasible and attractive.