Study of photorespiration in marine microalgae through the determination of glycolic using hydrophilic interaction liquid chromatography

Determination of organic s in intracellular extracts and in the cultivation media of marine microalgae aid investigations about metabolic routes related to assimilation of atmospheric carbon by these organisms, which are known by their role in the carbon dioxide sink. The separation of these s was investigated by hydrophilic interaction liquid chromatography (HILIC) using isocratic elution with a mobile phase composed of 70: 30 v/v acetonitrile/20 mmol/L ammonium acetate buffer (pH 6.8) and detection at 220 nm. HILIC allowed the determinations of glycolic , the most important metabolite for the evaluation of the photorespiration process in algae, to be made with better selectivity than that achieved by reversed phase liquid chromatography, but with less detectability. The concentration of glycolic was determined in the cultivation media and in intracellular extracts of the algae Tetraselmis gracilis and Phaeodactylum tricornutum submitted to different conditions of aeration: (i) without forced aeration, (ii) aeration with atmospheric air, and (iii) bubbling with N(2). The concentration of glycolic had a higher increase as the cultures were aerated with nitrogen, showing higher photorespiratory flux than that occurring in the cultures aerated with atmospheric air.

Curcumin-loaded into nanoparticles

The incorporation of the curcumin into poly(lactic-co-glycolic) () nanospheres by the nanoprecipitation technique, the characterization of the nanoparticles and the schistosomicidal activity of the curcumin-loaded into nanospheres were reported. The incorporation process occurred with high efficiency and the images of field-emission scanning electron microscopy (FESEM) revealed the production of spherically shaped particles. According to the dynamic light scattering measurements, the particles are nanometric and monodisperse. The curcumin-loaded nanoparticles (50 and 100 mu M) caused the death of all worms and a separation between 50% and 100% of Schistosoma mansoni couples at concentrations from 30 mu M. Moreover, the curcumin-loaded nanoparticles also decreased the motor activity and caused partial alterations in the tegument of adult worms. This study marks the first time that schistosomicidal activity has been reported for curcumin-loaded nanoparticles.

High-performance multifunctional graphene- fibers: toward biomimetic and conducting 3D scaffolds

The development of electrically conducting fibers based on known cytocompatible materials is of interest to those engaged in tissue regeneration using electrical stimulation. Herein, it is demonstrated that with the aid of rheological insights, optimized formulations of graphene containing spinnable poly(lactic-co-glycolic ) () dopes can be made possible. This helps extend the general understanding of the mechanics involved in order to deliberately translate the intrinsic superior electrical and mechanical properties of solution-processed graphene into the design process and practical fiber architectural engineering. The as-produced fibers are found to exhibit excellent electrical conductivity and electrochemical performance, good mechanical properties, and cellular affinity. At the highest loading of graphene (24.3 wt%), the conductivity of as-prepared fibers is as high as 150 S m-1 (more than two orders of magnitude higher than the highest conductivity achieved for any type of nanocarbon- composite fibers) reported previously. Moreover, the Young's modulus and tensile strength of the base fiber are enhanced 647- and 59-folds, respectively, through addition of graphene.

Tissue engineered prefabricated vascularized flaps

BACKGROUND.: Microvascular free tissue transfer has become increasingly popular in the reconstruction of head and neck defects, but it also has its disadvantages. Tissue engineering allows the generation of neo-tissue for implantation, but these tissues are often avascular. We propose to combine tissue-engineering techniques together with flap prefabrication techniques to generate a prefabricated vascularized soft tissue flap. METHODS: Human dermal fibroblasts (HDFs) labeled with fluorescein diacetate were static seeded onto polylactic-co-glycolic -collagen (-c) mesh. Controls were plain -c mesh. The femoral artery and vein of the nude rat was ligated and used as a vascular carrier for the constructs. After 4 weeks of implantation, the constructs were assessed by gross morphology, routine histology, Masson trichrome, and cell viability determined by green fluorescence. RESULTS: All the constructs maintained their initial shape and dimensions. Angiogenesis was evident in all the constructs with neo-capillary formation within the -c mesh seen. HDFs proliferated and filled the interyarn spaces of the -c mesh, while unseeded -c mesh remained relatively acellular. Cell tracer study indicated that the seeded HDFs remained viable and closely associated to remaining -c fibers. Collagen formation was more abundant in the constructs seeded with HDFs. CONCLUSIONS: -c, enveloped by a cell sheet composed of fibroblasts, can serve as a suitable scaffold for generation of a soft tissue flap. A ligated arteriovenous pedicle can serve as a vascular carrier for the generation of a tissue engineered vascularized flap.

Biomaterial scaffolds in cartilage-subchondral bone defects influencing the repair of autologous articular cartilage transplants

The repair of articular cartilage typically involves the repair of cartilage-subchondral bone tissue defects. Although various bioactive materials have been used to repair bone defects, how these bioactive materials in subchondral bone defects influence the repair of autologous cartilage transplant remains unclear. The aim of this study was to investigate the effects of different subchondral biomaterial scaffolds on the repair of autologous cartilage transplant in a sheep model. Cylindrical cartilage-subchondral bone defects were created in the right femoral knee joint of each sheep. The subchondral bone defects were implanted with hydroxyapatite-β-tricalcium phosphate (HA-TCP), poly lactic-glycolic ()-HA-TCP dual-layered composite scaffolds (/HA-TCP scaffolds), or autologous bone chips. The autologous cartilage layer was placed on top of the subchondral materials. After three months, the effect of different subchondral scaffolds on the repair of autologous cartilage transplant was systematically studied by investigating the mechanical strength, structural integration and histological responses. The results showed that the transplanted cartilage layer supported by HA-TCP scaffolds had better structural integration and higher mechanical strength than that supported by /HA-TCP scaffolds. Furthermore, HA-TCP supported cartilage showed higher expression of mucosubstances and glycol-amino-glycan (GAG) contents than that supported by /HA-TCP scaffolds. Our results suggested that the physicochemical properties, including the inherent mechanical strength and material chemistry of the scaffolds, play important roles in influencing the repair of autologous cartilage transplants. The study may provide useful information for the design and selection of proper subchondral biomaterials to support the repair of both subchondral bone and cartilage defects.

A simple method for fabricating 3-D multilayered composite scaffolds

One limitation of electrospinning stems from the charge build-up that occurs during processing, preventing further fibre deposition and limiting the scaffold overall thickness and hence their end-use in tissue engineering applications targeting large tissue defect repair. To overcome this, we have developed a technique in which thermally induced phase separation (TIPS) and electrospinning are combined. Thick three-dimensional, multilayered composite scaffolds were produced by simply stacking individual polycaprolactone (PCL) microfibrous electrospun discs into a cylindrical holder that was filled with a 3% poly(lactic-co-glycolic ) () solution in dimethylsulfoxide (a good solvent for but a poor one for PCL). The construct was quenched in liquid nitrogen and the solvent removed by leaching out in cold water. This technique enables the fabrication of scaffolds composed principally of electrospun membranes that have no limit to their thickness. The mechanical properties of these scaffolds were assessed under both quasi-static and dynamic conditions. The multilayered composite scaffolds had similar compressive properties to 5% PCL scaffolds fabricated solely by the TIPS methodology. However, tensile tests demonstrated that the multilayered construct outperformed a scaffold made purely by TIPS, highlighting the contribution of the electrospun component of the composite scaffold to enhancing the overall mechanical property slate. Cell studies revealed cell infiltration principally from the scaffold edges under static seeding conditions. This fabrication methodology permits the rapid construction of thick, strong scaffolds from a range of biodegradable polymers often used in tissue engineering, and will be particularly useful when large dimension electrospun scaffolds are required.

Controlling microencapsulation and release of micronized proteins using poly(ethylene glycol) and electrospraying

The fabrication of tailored microparticles for delivery of therapeutics is a challenge relying upon a complex interplay between processing parameters and materials properties. The emerging use of electrospraying allows better tailoring of particle morphologies and sizes than current techniques, critical to reproducible release profiles. While dry encapsulation of proteins is essential for the release of active therapeutics from microparticles, it is currently uncharacterized in electrospraying. To this end, poly(ethylene glycol) (PEG) was assessed as a micronizing and solubilizing agent for dry protein encapsulation and release from electrosprayed particles made from polycaprolactone (PCL). The physical effect of PEG in protein-loaded poly(lactic-co-glycolic ) () particles was also studied, for comparison. The addition of 5–15 wt% PEG 6 kDa or 35 kDa resulted in reduced PCL particle sizes and broadened distributions, which could be improved by tailoring the electrospraying processing parameters, namely by reducing polymer concentration and increasing flow rate. Upon micronization, protein particle size was reduced to the micrometer domain, resulting in homogenous encapsulation in electrosprayed PCL microparticles. Microparticle size distributions were shown to be the most determinant factor for protein release by diffusion and allowed specific control of release patterns.

Spontaneous large volume adipose tissue generation from a vascularized pedicled fat flap inside a chamber space

A novel method of spontaneous generation of new adipose tissue from an existing fat flap is described. A defined volume of fat flap based on the superficial inferior epigastric vascular pedicle in the rat was elevated and inset into a hollow plastic chamber implanted subcutaneously in the groin of the rat. The chamber walls were either perforated or solid and the chambers either contained poly(D,L-lactic-co-glycolic ) () sponge matrix or not. The contents were analyzed after being in situ for 6 weeks. The total volume of the flap tissue in all groups except the control groups, where the flap was not inserted into the chambers, increased significantly, especially in the perforated chambers (0.08 ± 0.007 mL baseline compared to 1.2 ± 0.08 mL in the intact ones). Volume analysis of individual component tissues within the flaps revealed that the adipocyte volume increased and was at a maximum in the chambers without , where it expanded from 0.04 ± 0.003 mL at insertion to 0.5 ± 0.08 mL (1250% increase) in the perforated chambers and to 0.16 ± 0.03 mL (400% increase) in the intact chambers. Addition of scaffolds resulted in less fat growth. Histomorphometric analysis rather than simple hypertrophy documented an increased number of adipocytes. The new tissue was highly vascularized and no fat necrosis or atypical changes were observed.

The influence of extracellular matrix on the generation of vascularized, engineered, transplantable tissue

In a recently described model for tissue engineering, an arteriovenous loop comprising the femoral artery and vein with interposed vein graft is fabricated in the groin of an adult male rat, placed inside a polycarbonate chamber, and incubated subcutaneously. New vascularized granulation tissue will generate on this loop for up to 12 weeks. In the study described in this paper three different extracellular matrices were investigated for their ability to accelerate the amount of tissue generated compared with a no-matrix control. Poly-D,L-lactic-co-glycolic () produced the maximal weight of new tissue and vascularization and this peaked at two weeks, but regressed by four weeks. Matrigel was next best. It peaked at four weeks but by eight weeks it also had regressed. Fibrin (20 and 80 mg/ml), by contrast, did not integrate with the generating vascularized tissue and produced less weight and volume of tissue than controls without matrix. The limiting factors to growth appear to be the chamber size and the capacity of the neotissue to integrate with the matrix. Once the sides of the chamber are reached or tissue fails to integrate, encapsulation and regression follow. The intrinsic position of the blood supply within the neotissue has many advantages for tissue and organ engineering, such as ability to seed the construct with stem cells and microsurgically transfer new tissue to another site within the individual. In conclusion, this study has found that and Matrigel are the best matrices for the rapid growth of new vascularized tissue suitable for replantation or transplantation.

Microparticle-mediated gene delivery for the enhanced expression of a 19-KDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP119) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic ) () microparticles of malaria DNA vaccines encoding MSP119 is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate microparticles at a flow rate of 18 mL h1. High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 lm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that -mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.

Novel surgical and imaging methods of the middle ear and temporal bone

Välikorvaleikkauksiin usein liittyvän välikorvan ja kuuloluuketjun kirurgisen rekonstruktion tavoitteena on luoda olosuhteet, jotka mahdollistavat hyvän kuulon sekä välikorvan säilymisen tulehduksettomana ja ilmapitoisena. Välikorvan rekonstruktiossa on käytetty implanttimateriaaleina perinteisesti potilaan omia kudoksia sekä tarvittaessa erilaisia hajoamattomia biomateriaaleja, mm. titaania ja silikonia. Ongelmana biomateriaalien käytössä voi olla bakteerien adherenssi eli tarttuminen vieraan materiaalin pintaan, mikä saattaa johtaa biofilmin muodostumiseen. Tämä voi aiheuttaa kroonisen, huonosti antibiootteihin reagoivan infektion kudoksessa, mikä usein käytännössä johtaa uusintaleikkaukseen ja implantin poistoon. Maitohappo- ja glykolihappopohjaiset biologisesti hajoavat polymeerit ovat olleet kliinisessä käytössä jo vuosikymmeniä. Niitä on käytetty erityisesti tukimateriaaleina mm. ortopediassa sekä kasvo- ja leukakirurgiassa. Niitä ei ole toistaiseksi käytetty välikorvakirurgiassa. Korvan kuvantamiseen käytetään ensisijaisesti tietokonetomografiaa (TT). TT-tutkimuksen ongelmana on potilaan altistuminen suhteellisen korkealle sädeannokselle, joka kasvaa kumulatiivisesti, jos kuvaus joudutaan toistamaan. Väitöskirjatyö selvittää uuden, aiemmin kliinisessä työssä rutiinisti lähinnä hampaiston ja kasvojen alueen kuvantamiseen käytetyn rajoitetun kartiokeila-TT:n soveltuvuutta korvan alueen kuvantamiseen. Väitöskirjan kahdessa ensimmäisessä osatyössä tutkittiin ja verrattiin kahden kroonisia ja postoperatiivisia korvainfektioita aiheuttavan bakteerin, Staphylococcus aureuksen ja Pseudomonas aeruginosan, in vitro adherenssia titaanin, silikonin ja kahden eri biohajoavan polymeerin () pintaan. Lisäksi tutkittiin materiaalien albumiinipinnoituksen vaikutusta adherenssiin. Kolmannessa osatyössä tutkittiin eläinmallissa :n biokompatibiliteettia eli kudosyhteensopivuutta kokeellisessa välikorvakirurgiassa. Chinchillojen välikorviin istutettiin -materiaalia, eläimiä seurattiin, ja ne lopetettiin 6 kk:n kuluttua operaatiosta. Biokompatibiliteetin arviointi perustui kliinisiin havaintoihin sekä kudosnäytteisiin. Neljännessä osatyössä tutkittiin kartiokeila-TT:n soveltuvuutta korvan alueen kuvantamiseen vertaamalla sen tarkkuutta perinteisen spiraali-TT:n tarkkuuteen. Molemmilla laitteilla kuvattiin ohimo- eli temporaaliluita korvan alueen kliinisesti ja kirurgisesti tärkeiden rakenteiden kuvantumisen tarkkuuden arvioimiseksi. Viidennessä osatyössä arvioitiin myös operoitujen temporaaliluiden kuvantumista kartiokeila-TT:ssa. Bakteeritutkimuksissa -materiaalin pintaan tarttui keskimäärin korkeintaan saman verran tai vähemmän bakteereita kuin silikonin tai titaanin. Albumiinipinnoitus vähensi bakteeriadherenssia merkitsevästi kaikilla materiaaleilla. Eläinkokeiden perusteella todettiin hyvin siedetyksi välikorvassa. Korvakäytävissä tai välikorvissa ei todettu infektioita, tärykalvon perforaatioita tai materiaalin esiin työntymistä. Kudosnäytteissä näkyi lievää tulehdusreaktiota ja fibroosia implantin ympärillä. Temporaaliluutöissä rajoitettu kartiokeila-TT todettiin vähintään yhtä tarkaksi menetelmäksi kuin spiraali-TT välikorvan ja sisäkorvan rakenteiden kuvantamisessa, ja sen aiheuttama kertasäderasitus todettiin spiraali-TT:n vastaavaa huomattavasti vähäisemmäksi. Kartiokeila-TT soveltui hyvin välikorvaimplanttien ja postoperatiivisen korvan kuvantamiseen. Tulokset osoittavat, että on välikorvakirurgiaan soveltuva, turvallinen ja kudosyhteensopiva biomateriaali. Biomateriaalien pinnoittaminen albumiinilla vähentää merkittävästi bakteeriadherenssia niihin, mikä puoltaa pinnoituksen soveltamista implanttikirurgiassa. Kartiokeila-TT soveltuu korvan alueen kuvantamiseen. Sen tarkkuus kliinisesti tärkeiden rakenteiden osoittamisessa on vähintään yhtä hyvä ja sen potilaalle aiheuttama sädeannos pienempi kuin nykyisen korva-spiraali-TT:n. Tämä tekee menetelmästä spiraali-TT:aa potilasturvallisemman vaihtoehdon erityisesti, jos potilaan tilanne vaatii seurantaa ja useampia kuvauksia, ja jos halutaan kuvata rajoitettuja alueita uni- tai bilateraalisesti.

Co-electrospun poly(lactide-co-glycolide), gelatin, and elastin blends for tissue engineering scaffolds

In this study, we describe composite scaffolds composed of synthetic and natural materials with physicochemical properties suitable for tissue engineering applications. Fibrous scaffolds were co-electrospun from a blend of a synthetic biodegradable polymer (poly(lactic-co-glycolic ), , 10% solution) and two natural proteins, gelatin (denatured collagen, 8% solution) and (x-elastin (20% solution) at ratios of 3:1:2 and 2:2:2 (v/v/v). The resulting -gelatin-elastin (PGE) fibers were homogeneous in appearance with an average diameter of 380 80 mn, which was considerably smaller than fibers made under identical conditions from the starting materials (, 780 +/- 200 nm; gelatin, 447 +/- 1.23 nm; elastin, 1060 170 nm). Upon hydration, PGE fibers swelled to an average fiber diameter of 963 +/- 132 nm, but did not disintegrate. Importantly, PGE scaffolds were stable in an aqueous environment without crosslinking, and were more elastic than those made of pure elastin fibers. To investigate the cytocompatibility of PGE, we cultured H9c2 rat cardiac myoblasts and rat bone marrow stromal cells (BMSCs) on fibrous PGE scaffolds. We found that myoblasts grew equally as well or slightly better on the scaffolds than on tissue-culture plastic. Microscopic evaluation confirmed that myoblasts reached confluence on the scaffold surfaces while simultaneously growing into the scaffolds.

The potential of electron beam radiation for simultaneous surface modification and bioresorption control of PLLA

Bioresorbable polymers have been widely investigated as materials exhibiting significant potential for successful application in the fields of tissue engineering and drug delivery. Further to the ability to control degradation, surface engineering of polymers has been highlighted as a key method central to their development. Previous work has demonstrated the ability of electron beam (e-beam) technology to control the degradation profiles and bioresorption of a number of commercially relevant bioresorbable polymers (poly-l-lactic (PLLA), Llactide/DL-lactide co-polymer (PLDL) and poly(lactic-co-glycolic ()). This work investigates the further potential of ebeam technology to impart added biofunctionality through the manipulation of polymer (PLLA) surface properties. PLLA samples were subjected to e-beam treatments in air, with varying beam energies and doses. Surface characterization was then performed using contact angle analysis, X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and atomic force microscopy. Results demonstrated a significant increase in surface wettability post e-beam treatment. In correlation with this, XPS data showed the introduction of oxygen-containing functional groups to the surface of PLLA. Raman spectroscopy indicated chain scission in the near surface region of PLLA (as predicted). However, e-beam effects on surface properties were not shown to be dependent on beam energy or dose. E-beam irradiation did not seem to affect the surface roughness of PLLA as a direct consequence of the treatment.

Microneedle/nanoencapsulation-mediated transdermal delivery:Mechanistic insights

A systematic study was undertaken to gain more insight into the mechanism of transdermal delivery of nanoencapsulated model dyes across microneedle (MN)-treated skin, a complex process not yet explored. Rhodamine B (Rh B) and fluorescein isothiocyanate (FITC) as model hydrophilic and hydrophobic small/medium-size molecules, respectively, were encapsulated in poly lactic-co-glycolic () nanoparticles (NPs) and delivered through full thickness porcine skin pretreated with MN array. Permeation through MN-treated skin was affected by physicochemical characteristics of NPs and the encapsulated dyes. Dye flux was enhanced by smaller particle size, hydrophilicity, and negative zeta potential of NPs. Regarding encapsulated dyes, solubility at physiological pH and potential interaction with skin proteins proved to outweigh molecular weight as determinants of skin permeation. Data were verified using confocal laser scanning microscopy imaging. Findings coupled with the literature data are supportive of a mechanism involving influx of NPs, particularly of smaller size, deep into MN-created channels, generating depot dye-rich reservoirs. Molecular diffusion of the released dye across viable skin layers proceeds at a rate determined by its molecular characteristics. Data obtained provide mechanistic information of importance to the development of formulation strategies for more effective intradermal and transdermal MN-mediated delivery of nanoencapsulated therapeutic agents.