Strain-Stiffening of Agarose Gels

Strain-stiffening is one of the characteristic properties of biological hydrogels and extracellular matrices, where the stiffness increases upon increased deformation. Whereas strain-stiffening is ubiquitous in protein-based materials, it has been less observed for polysaccharide and synthetic polymer gels. Here we show that agarose, that is, a common linear polysaccharide, forms helical fibrillar bundles upon cooling from aqueous solution. The hydrogels with these semiflexible fibrils show pronounced strain-stiffening. However, to reveal strain-stiffening, suppressing wall slippage turned as untrivial. Upon exploring different sample preparation techniques and rheological architectures, the cross-hatched parallel plate geometries and in situ gelation in the rheometer successfully prevented the slippage and resolved the strain-stiffening behavior. Combining with microscopy, we conclude that strain-stiffening is due to the semiflexible nature of the agarose fibrils and their geometrical connectivity, which is below the central-force isostatic critical connectivity. The biocompatibility and the observed strain-stiffening suggest the potential of agarose hydrogels in biomedical applications.Peer reviewe

Rapid Self-Healing and Thixotropic Organogelation of Amphiphilic Oleanolic Acid-Spermine Conjugates

Natural and abundant plant triterpenoids are attractive starting materials for the synthesis of conformationally rigid and chiral building blocks for functional soft materials. Here, we report the rational design of three oleanolic acid-triazole-spermine conjugates, containing either one or two spermine units in the target molecules, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. The resulting amphiphile-like molecules 2 and 3, bearing just one spermine unit in the respective molecules, self-assemble into highly entangled fibrous networks leading to gelation at a concentration as low as 0.5% in alcoholic solvents. Using step-strain rheological measurements, we show rapid self-recovery (up to 96% of the initial storage modulus) and sol ⇔ gel transition under several cycles. Interestingly, rheological flow curves reveal the thixotropic behavior of the gels. To the best of our knowledge, this kind of behavior was not shown in the literature before, neither for a triterpenoid nor for its derivatives. Conjugate 4, having a bolaamphiphile-like structure, was found to be a nongelator. Our results indicate that the position and number of spermine units alter the gelation properties, gel strength, and their self-assembly behavior. Preliminary cytotoxicity studies of the target compounds 2-4 in four human cancer cell lines suggest that the position and number of spermine units affect the biological activity. Our results also encourage exploring other triterpenoids and their derivatives as sustainable, renewable, and biologically active building blocks for multifunctional soft organic nanomaterials.acceptedVersionPeer reviewe

Compressive stress-mediated p38 activation required for ER alpha plus phenotype in breast cancer

Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ER alpha + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ER alpha-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ER alpha + breast cancer models. The ER alpha + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ER alpha is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ER alpha signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ER alpha phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK. Reliable luminal estrogen receptor (ER alpha+) breast cancer models are limited. Here, the authors use patient derived breast epithelial and breast cancer explant cultures grown in several extracellular matrix scaffolds and show that ER alpha expression is regulated by matrix stiffness via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation.Peer reviewe

Compressive stress-mediated p38 activation required for ERα + phenotype in breast cancer

Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ER alpha + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ER alpha-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ER alpha + breast cancer models. The ER alpha + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ER alpha is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ER alpha signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ER alpha phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK.Reliable luminal estrogen receptor (ER alpha+) breast cancer models are limited. Here, the authors use patient derived breast epithelial and breast cancer explant cultures grown in several extracellular matrix scaffolds and show that ER alpha expression is regulated by matrix stiffness via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation.</p

Polymeeri- ja geeliverkot

Nature has created materials with extraordinary functional properties, based on complex and hierarchical structures over multiple length scales. Mechanical properties are among the most interesting features, such as strain-stiffening of biopolymer networks. Using this as a source of inspiration, self-assembly has been used to tailor structures from nanoscale to macroscale objects. This dissertation focuses on controlled self-assemblies, structures, properties, and finally, a biomedical application. In Publication I, amphiphilic star-shaped polymers with supramolecularly directing units were synthesized to obtain hierarchical assemblies. Selection of the solvent triggered hierarchical self-assembly from nanometric micelles to micrometric spherical structures. In Publication II, the thermo- and UV-responsive gelation of a copolymer and cellulose nanocrystals (CNCs) was designed. This sequential approach offered a tool for homogeneous mixing and subsequent in-situ gelation based on light triggered opposite charging. Reversible or irreversible gelation could be achieved according to the ratio of the components and heating time. Publications III-IV show strain-stiffening and negative normal force of agarose hydrogels using two rheological protocols. Strain-stiffening was observed when slipping was suppressed. Strain-stiffening was analyzed providing an explanation of the origin of the behavior. The negative normal force of the agarose hydrogels was displayed. In Publication V, various hydrogels as 3D matrices for patient-derived breast cancer tissue explant culturing (PDEC) was investigated. The chemical composition of the matrix regulates the luminal and basal cell identity. The stiffness of the matrix regulates the expression of the estrogen hormone receptor (ERα). In conclusion, controlled network self-assembly facilitates the production of functional materials. In particular, network properties can help to develop cell culturing platforms for breast cancer research and synthetic strain-stiffening materials.Luonnossa on funktionaalisia materiaaleja, joiden toiminta ja ominaisuudet ovat seurausta monitasoisesta järjestymisestä materiaalin rakenteessa. Eräs esimerkki luonnon materiaalien erityisistä mekaanisista ominaisuuksista on biopolymeeriverkkojen myötöjäykistyminen. Materiaaliteknologia pyrkii jäljittelemään näitä ominaisuuksia hyödyntämällä molekyylien kontrolloitua itsejärjestymistä. Tässä väitöskirjassa käsitellään verkkorakenteiden kontrolloitua itsejärjestymistä, rakenteita, ominaisuuksia sekä käyttöä biolääketieteellisessä sovelluksessa. Julkaisussa I syntetisoitiin tähtimäinen amfifiilinen polymeeri, jonka supramolekulaarista itsejärjestymistä tutkittiin. Liuottimen vaihdolla aikaansaatiin monitasoisia rakenteita, nanometrin kokoisista miselleistä mikrokokoisiin pallomaisiin rakenteisiin. Julkaisussa II suunniteltiin kopolymeerin ja selluloosan nanokiteiden (CNC) kaksivaiheinen geeliytyminen. Ensimmäisessä vaiheessa anionisen selluloosan vesidispersioon sekoitettiin neutraalia polymeeriä, jonka jälkeen sen kationinen varaus saatiin aikaiseksi UV-säteilyllä. Vastakkaisten varausten aikaansaamaa geeliytymistä ja sen palautumista pystyttiin säätelemään lämmitysajan sekä polymeerin ja CNC:n suhteellisten määrien avulla. Julkaisuissa III-IV tutkittiin agaroosihydrogeelin myötöjäykistymistä vertailemalla kahta eri reologista mittausmenetelmää. Myötöjäykistyminen pystyttiin toteamaan, kun geelin lipsuminen mittauksen aikana estettiin. Myötöjäykistymisen syntymekanismia analysoitiin sekä esitettiin agaroosigeelin negativinen normaalivoima. Julkaisussa V hyödynnettiin erilaisia hydrogeelejä 3D-solukasvastusalustoina potilasperäisille rintasyövän kudoseksplanteille. Geelien kemiallinen koostumus vaikutti luminaalisten solutyyppien muuttumiseen basaalisoluiksi. Lisäksi geelien jäykkyys sääteli estrogeenireseptorin ilmeentymistä luminaalisissa soluissa. Väitöskirjassa tutkittiin verkkorakanteiden itsejärjestymisen säätelyyn liittyviä työkaluja, joita voidaan hyödyntää toiminnallisten materiaalien valmistamisessa. Lisäksi väitöskirjassa tutkittiin hydrogeelien verkkorakenteiden ominaisuuksia, joita voidaan hyödyntää synteettisten myötöjäykistyvien materiaalien sekä rintasyöpäsolujen 3D-kasvatusalustojen kehittämisessä

Salaatti- ja tupakkanäytteiden homogenisointimenetelmien vertailu DNA-puhdistukseen magneettipartikkeliprosessorilla

Magneettipartikkelitekniikkaan perustuvat magneettipartikkeliprosessorit on kehitetty erilaisten molekyylien kuten nukleiinihappojen puhdistuksen automatisointiin. Magneettipartikkeliprosessorit eristävät korkeasaantoista, puhdasta ja laadukasta DNA:ta ja RNA:ta erilaisista näytemateriaaleista. Työn tavoitteena oli vertailla erilaisia menetelmiä salaatti- ja tupakkanäytteiden homogenisoimiseksi DNA:n eristämiseen magneettipartikkeliprosessorilla. Homogenisointimenetelmiksi valittiin mortteli ja survin, Talboys-kuulamylly, Pellet Pestle -mikrosentrifugiputkisekoittaja, Ulta-Turrax-koeputkisekoittaja ja Ribolyser-homogenisaattori. Lisäksi homogenisointimenetelmillä testattiin neljää erilaista homogenisointiolosuhdetta: näytteen homogenisointia sellaisenaan, homogenisointia lyysispuskurissa, homogenisointia nestetypessä jäädytettynä ja kahden viimeiseksi mainitun yhdistelmätekniikkaa. Eristetyn DNA:n määrää, puhtautta ja laatua arvioitiin absorbanssimittauksilla, agaroosigeelielektroforeesilla ja PCR:llä. Tuloksien perusteella korkeasaantoisimmat, puhtaimmat, laadukkaimmat ja toistettavimmat lopputuotteet saatiin eristettyä morttelilla, Ribolyser-homogenisaattorilla ja Talboys-kuulamyllyllä. Homogenisointiolosuhteista parhaimmaksi osoittautui tuloksien perusteella näytteen homogenisoiminen lyysispuskurissa. Talboys-kuulamyllyn todettiin olevan työssä käytetyistä homogenisointimenetelmistä ainoa varteen otettava vaihtoehto, jos halutaan homogenisoida paljon näytteitä samanaikaisesti. Morttelin taas todettiin olevan erityisen hyvä vaihtoehto, jos näytteitä on lukumäärällisesti vähän tai ne voidaan homogenisoida yhdessä. Työssä saatiin laaja kuva erilaisten homogenisointimenetelmien ja –olosuhteiden ominaisuuksista ja käytettävyyksistä salaatti- ja tupakkanäytteille. Työn avulla pystytään suosittelemaan ja neuvomaan asiakkaita löytämään juuri heidän tarkoituksiinsa sopiva homogenisointimenetelmä kasvinäytteille, jota aikaisemmin ei ole pystytty tekemään.Magnetic bead based magnetic particle processors have been developed to automate to purify various molecules such as nucleic acid. Magnetic particle processors isolate high-yield, purity and high-quality DNA and RNA from various sample materials. The aim of this thesis project was to compare different methods of homogenizing lettuce and tobacco for DNA purification with a magnetic particle processor. The homogenization methods selected for this project were a mortar and pestle, a Talboys ball mill, a Pellet Pestle microcentrifuge tube mixer, an Ulta-Turrax test tube mixer and a Ribolyser homogenizer. In addition, four different types of circumstances of homogenization were tested: homogenization, homogenization with a lysis buffer, homogenization with liquid nitrogen and homogenization with a lysis buffer and liquid nitrogen. The amount, purity and quality of isolated DNA were evaluated by measurement of absorbance and by agarose gel electroforesis and PCR. The results of this project showed that the highest yield, the purest, highest quality and the most reproducible results were provided by the mortar and pestle, the Ribolyser homogenizer and the Talboys ball mill. According to the results, homogenization with a lysis buffer was an optimal circumstance for DNA isolating from lettuce and tobacco. The Talboys ball mill was found to be the only viable option to homogenize several samples simultaneously. Where as the mortar and pestle were found to be an extremely viable homogenization option for few samples or samples which can be homogenized together. Project provided a wide insight into the properties and usability of different homogenization methods and circumstances for lettuce and tobacco samples. On the basis of the results of the project, recommendations can be made and advice can be provided to help clients find the suitable homogenization method for their purpose, which previously has not been possible

Strain Stiffening and Negative Normal Force of Agarose Hydrogels

| openaire: EC/H2020/742829/EU//DRIVENInspired by the specific strain stiffening and negative normal force phenomena in several biological networks, herein, we show strain stiffening and negative normal force in agarose hydrogels. We use both pre-strain and strain amplitude sweep protocols in dynamic rheological measurements where the gel slip was suppressed by the in situ gelation in the cross-hatched parallel plate rheometer geometry. Within the stiffening region, we show the scaling relation for the differential modulus K ∝ σ1, where σ is stress. The strain at the onset of stiffening is almost constant throughout the concentration range. The gels show negative apparent normal stress difference when sheared as a result of the gel contraction. The pore size of the hydrogel is large enough to allow water to move with respect to the network to balance the pressure difference caused by the hoop stress. The rheological analysis together with scanning electron microscopy suggests that the agarose gels can be described using subisostatic athermal network models where the connectivity dictates the stiffening behavior. Therefore, the simple agarose gels appear to capture several of the viscoelastic properties, which were previously thought to be characteristic to biological protein macromolecules.Peer reviewe

Bipyridine based metallogels : an unprecedented difference in photochemical and chemical reduction in the in situ nanoparticle formation

Metal co-ordination induced supramolecular gelation of low molecular weight organic ligands is a rapidly expanding area of research due to the potential in creating hierarchically self-assembled multi-stimuli responsive materials. In this context, structurally simple O-methylpyridine derivatives of 4,4′-dihydroxy-2,2′-bipyridine ligands are reported. Upon complexation with Ag(I) ions in aqueous dimethyl sulfoxide (DMSO) solutions the ligands spontaneously form metallosupramolecular gels at concentrations as low as 0.6 w/v%. The metal ions induce the self-assembly of three dimensional (3D) fibrillar networks followed by the spontaneous in situ reduction of the Ag-centers to silver nanoparticles (AgNPs) when exposed to daylight. Significant size and morphological differences of the AgNP's was observed between the standard chemical and photochemical reduction of the metallogels. The gelation ability, the nanoparticle formation and rheological properties were found to be depend on the ligand structure, while the strength of the gels is affected by the water content of the gels.peerReviewe

UV-Triggered On-Demand Temperature-Responsive Reversible and Irreversible Gelation of Cellulose Nanocrystals

We show ionically cross-linked, temperature-responsive reversible or irreversible hydrogels of anionic cellulose nanocrystals (CNCs) and methacrylate terpolymers by mixing them homogeneously in the initially charge-neutral state of the polymer, which was subsequently switched to be cationic by cleaving side groups by UV irradiation. The polymer is a random terpolymer poly(di(ethylene glycol) methyl ether methacrylate)-rnd-poly(oligo(ethylene glycol) methyl ether methacrylate)-rnd-poly(2-((2-nitrobenzyl)oxycarbonyl)aminoethyl methacrylate), that is, PDEGMA-rnd-POEGMA-rnd-PNBOCAEMA. The PDEGMA and POEGMA repeating units lead to a lower critical solution temperature (LCST) behavior. Initially, homogeneous aqueous mixtures are obtained with CNCs, and no gelation is observed even upon heating to 60 °C. However, upon UV irradiation, the NBOCAEMAs are transformed to cationic 2-aminoethyl methacrylate (AEMA) groups, as 2-nitrobenzaldehyde moieties are cleaved. The resulting mixtures of anionic CNC and cationic PDEGMA-rnd-POEGMA-rnd-PAEMA show gelation for sufficiently high polymer fractions upon heating to 60 °C due to the interplay of ionic interactions and LCST. For short heating times, the gelation is thermoreversible, whereas for long enough heating times, irreversible gels can be obtained, indicating importance of kinetic aspects. The ionic nature of the cross-linking is directly shown by adding NaCl, which leads to gel melting. In conclusion, the optical triggering of the polymer ionic interactions in combination with its LCST phase behavior allows a new way for ionic nanocellulose hydrogel assemblies.Peer reviewe