Temporal and Spatial Nanobiomaterials for Tissue Engineering and Drug Delivery

The intersection of materials science, biology, and nanotechnology has allowed for the development of advanced nanobiomaterials for tissue engineering and drug delivery. With more knowledge of how physical and chemical properties of a biomaterial influence cell function and response, it is important to impart different characteristics to materials with which cells will interact. Characteristics to consider for tissue engineering and/or drug delivery applications include: biocompatibility, mechanical properties, surface area, and ligand presentation. As foreign materials that are placed into the body and are not necessary permanently, these materials should also be biodegradable. Previous biomaterials have fallen short in many ways (i.e. lack of degradability, poor modulus matching, lack of porosity), as it is difficult to design a material with all necessary attributes. The more biomimetic and tailorable a material is, the better suited it is for these applications. New chemistries and approaches must be considered to incorporate all necessary characteristics. This work introduces two new materials that are characterized and evaluated for biomaterials applications and successfully overcome the temporal and spatial shortcomings of previous research. 2-methylene-1,3,6-trioxocane (MTC) is a hydrophobic monomer that is crosslinked with poly(ethylene glycol) diacrylate, a hydrophilic crosslinker, at varying crosslinker concentrations and molecular weights. In this work, with respect to tissue engineering, the materials’ morphological changes, swelling, degradation, and elastic modulus properties are all assessed. Tunability is found in these properties as the crosslinker is adjusted and a hydrophobic-hydrophilic balance dictates many behavioral properties, including an atypical increase in swelling as crosslinker concentration is increased. The biocompatibility is assessed with MTC formulations with 575 Da and 2 kDa crosslinker at 1.0% crosslinker concentrations exhibiting moderate swelling (< 100%) and modulus of ~100 kPa showing good biocompatibility and utility for soft tissue engineering applications. As a drug delivery system (DDS), crosslinked MTC samples were evaluated in terms of tunability and kinetics of drug release behavior. Drug release was tested for three different types of drugs: small molecule hydrophobic, small molecule hydrophilic, and a protein. From the 10-week studies, MTC hydrogels importantly demonstrated suitability for controlled release of the small molecule hydrophobic drug, with constant zero order kinetics displayed across crosslinker variations at physiological pH; the model protein, exhibited first order behavior and increasing drug release as crosslinker concentration increases. Rapid, 1 minute subcutaneous in situ gelling was also demonstrated in a mouse, making MTC advantageous as an injectable DDS. Finally, polymeric nanoparticle functionalization is explored to improve drug targeting/internalization to treat HER2+ breast cancer. This strategy is tested by (1) comparing nanoparticles fabricated from a linear PEGMA-PLGA copolymer versus a novel palm-tree PEGPET-PLGA copolymer chemistry and (2) testing a new targeting peptide sequence and its modified targeting-internalization sequences through the addition of the TAT cell-penetrating peptide sequence. Through early experiments, more than 2 times the binding affinity was measured in vitro for multi-functionalized nanoparticles compared to linear nanoparticles, showing that the increased peptide presentation on the nanoparticles’ surface fabricated with the PEGPET-PLGA copolymer helps enhance cell targeting. Select combination peptide sequences with TAT also show evidence of increased HER2+ affinity. From this thesis, contributions are made to the field of biomaterials by not only providing new materials and chemistries available for varied biomaterials use, but most critically, provides commentary on their necessity, methods to modulate these nanobiomaterials temporally and spatially and appropriate characterization, and their ongoing use.PHDMacromolecular Science & EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163136/1/doleyres_1.pd

Production en continu de ferments lactiques probiotiques par la technologie des cellules immobilisées

Pour produire des ferments lactiques contenant des bifidobactéries par des cellules immobilisées, des fermentations ont été réalisées avec une culture pure de bifidobactéries ou en culture mixte avec une souche de lactocoques. Une méthode a été développée pour localiser et quantifier par immunofluorescence les deux souches dans des billes de gel. L'utilisation de cette méthode a permis d'observer une croissance bactérienne préférentielle à la périphérie des billes. La production continue de culture mixte a été étudiée avec un système composé d'un premier réacteur (R1) contenant les deux souches immobilisées séparément dans des billes de gel et d'un second réacteur (R2) opéré avec les cellules libres relarguées de R1. Une culture mixte concentrée de composition stable a été produite à 35ʻC dans l'effluent de R2. La redistribution des souches dans les billes fut observée en microscopie confocale. La résistance à différents stress des cellules libres produites dans l'effluent des deux réacteurs a augmenté avec le temps de fermentation

Production of 3-hydroxypropionaldehyde using a two-step process with Lactobacillus reuteri

3-Hydroxypropionaldehyde (3-HPA) produced by Lactobacillus reuteri is a broad-spectrum antimicrobial substance of glycerol conversion. The aim of the present work was to optimize 3-HPA production by Lb. reuteri ATCC 53608 using a two-step process. The first step was the production of Lb. reuteri cells in optimal conditions. Cells were then harvested by centrifugation and suspended in glycerol solution, which the resting cells bioconverted to 3-HPA. The effect of biomass concentration, temperature, glycerol concentration, anaerobic/micro-aerophilic conditions, and incubation time was studied for high 3-HPA production. 3-HPA accumulation was limited by the death of cells in contact with high concentrations of 3-HPA. However, a very high 3-HPA concentration of 235±3mM was obtained after 45min of incubation at 30°C in 400mM glycerol for an initial free-cell concentration of 1.6±0.3×1010 viablecells/ml. A high viability was maintained at low temperatures in the range 5-15°C, but with a slightly lower yield of 3-HPA at 5°C compared with higher temperatures, up to 37°C. Successive 1-h incubations of Lb. reuteri cells in 200mM glycerol at 15°C to tentatively reuse the cells resulted in decreasing 3-HPA concentrations at the end of each cycle, with two successful production cycles yielding high 3-HPA concentrations of 147±1mM and 128±2m

Replacement and deletion mutations in the catalytic domain and belt region of Aspergillus awamori glucoamylase to enhance thermostability

Three single-residue mutations, Asp71→Asn, Gln409→Pro and Gly447→Ser, two long-to-short loop replacement mutations, Gly23-Ala24-Asp25-Gly26-Ala27-Trp28-Val29-Ser30→Asn-Pro-Pro (23–30 replacement) and Asp297-Ser298-Glu299-Ala300-Val301→Ala-Gly-Ala (297–301 replacement) and one deletion mutation removing Glu439, Thr440 and Ser441 (Δ439–441), all based on amino acid sequence alignments, were made to improve Aspergillus awamori glucoamylase thermostability. The first and second single-residue mutations were designed to introduce a potential N-glycosylation site and to restrict backbone bond rotation, respectively, and therefore to decrease entropy during protein unfolding. The third single-residue mutation was made to decrease flexibility and increase O-glycosylation in the already highly O-glycosylated belt region that extends around the globular catalytic domain. The 23–30 replacement mutation was designed to eliminate a very thermolabile extended loop on the catalytic domain surface and to bring the remainder of this region closer to the rest of the catalytic domain, therefore preventing it from unfolding. The 297–301 replacement mutant GA was made to understand the function of the random coil region between α-helices 9 and 10. Δ439–441 was constructed to decrease belt flexibility. All six mutations increased glucoamylase thermostability without significantly changing enzyme kinetic properties, with the 23–30 replacement mutation increasing the activation free energy for thermoinactivation by about 4 kJ/mol, which leads to a 4°C increase in operating temperature at constant thermostability

Adherence measurements and corrosion resistance in primer/hot-dip galvanized steel systems

This paper focuses on the adherence during ageing of a primer (made of polyester resins crosslinked with melamine) applied onto hot-dip galvanized (HDG) steel for coil coating application and its influence on corrosion protection. A chromium-free surface treatment, composed of fluorotitanic acid, phosphoric acid, manganese phosphate, and vinylphenol was applied on the HDG steel to obtain high corrosion resistance and high adherence of a polyester and melamine primer. The influence of the manganese phosphate on the corrosion and adherence was investigated. To measure the adherence between the metal and the primer, a three-point flexure test was set up. The adherence was then linked with corrosion resistance during ageing, using electrochemical impedance spectroscopy

Production of 3-hydroxypropionaldehyde using a two-step process with Lactobacillus reuteri

ISSN:0175-7598ISSN:1432-061