Cell Microenvironment Engineering and Monitoring for Tissue Engineering and Regenerative Medicine: The Recent Advances

In tissue engineering and regenerative medicine, the conditions in the immediate vicinity of the cells have a direct effect on cells' behaviour and subsequently on clinical outcomes. Physical, chemical, and biological control of cell microenvironment are of crucial importance for the ability to direct and control cell behaviour in 3-dimensional tissue engineering scaffolds spatially and temporally. In this review, we will focus on the different aspects of cell microenvironment such as surface micro-, nanotopography, extracellular matrix composition and distribution, controlled release of soluble factors, and mechanical stress/strain conditions and how these aspects and their interactions can be used to achieve a higher degree of control over cellular activities. The effect of these parameters on the cellular behaviour within tissue engineering context is discussed and how these parameters are used to develop engineered tissues is elaborated. Also, recent techniques developed for the monitoring of the cell microenvironment in vitro and in vivo are reviewed, together with recent tissue engineering applications where the control of cell microenvironment has been exploited. Cell microenvironment engineering and monitoring are crucial parts of tissue engineering efforts and systems which utilize different components of the cell microenvironment simultaneously can provide more functional engineered tissues in the near future

Mikro ve nanodesenli polimerik yüzeylerle farklı hücre tipleri arasındaki iletişim.

Micro and nanopatterned surfaces are powerful experimental platforms for investigating the mechanisms of cell adhesion, cell orientation, differentiation and they enable significant contributions to the fields of basic cell and stem cell biology, and tissue engineering. In this study, interaction between micro and nanopatterned polymeric surfaces and different cell types was investigated. Three types of micropillars were produced by photolithography (Type 1-3), while nanometer sized pillars were produced in the form of an array by electron beam lithography (EBL). Replica of silicon masters were made of polydimethylsiloxane (PDMS). Polymeric [P(L-D,L)LA and a P(L-D,L)LA:PLGA blend] replica were prepared by solvent casting of these on the PDMS template and used in in vitro studies. The final substrates were characterized by various microscopic methods such as light microscopy, atomic force microscopy (AFM) and scanning electron microscopy (SEM). In order to investigate deformation of the nucleus in response to the physical restrictions imposed by micropillars, Type 1 and Type 2 pillars were used. These substrates were covered with pillars with different interpillar distances. While Type 1 is covered with symmetrically (in X-Y directions) distributed pillars, Type 2 pillars were distributed asymmetrically and the inter-pillar distances were increased. Nuclei deformation of five cell v types, two cancer cell lines (MCF7 and Saos-2), one healthy bone cell (hFOB1.19), one stem cell (bone marrow origined mesemchymal stem cells, BMSCs) and one standard biomaterial test cell type, (L929) fibroblasts was examined by using fluorescence microscopy and SEM. The nuclei of Saos-2 and MCF7 cells were found to be deformed most drastically. Nucleus deformation and intactness of nuclear membrane was examined by Anti- Lamin A staining. The interaction of the cells with micropillars was visualized by labelling focal adhesion complexes (FAC). Wettabilities of patterned and smooth surfaces were determined. As the patterns become denser (closer micropillars, Type 1) the hydrophobicity increased. Similar to water droplets, the cells were mostly spread at the top of the Type 1 pillars. The number of cells spread on the substrate surface was much higher on Type 2 patterned films. In order to support these qualitative findings, nucleus deformation was quantified by image analysis. Frequency of nucleus deformation was determined as the ratio of deformed to the total number of nuclei (%). In order to quantify the intensity of nuclei deformation, their circularity was evaluated. In addition to nucleus deformation, alterations in the ratio of cell area-to-nucleus area in response to micropillars were determined by image analysis. The results indicated that cancerous cells were more deformable. The qualitative microscopic evaluation and the data obtained by quantification of the nucleus and cellular deformation were in good agreement. In addition, the findings were consistent with expectations which suggest that cancerous cells are “softer”. In the second part of the research the force applied by the cells on arrays of micropillars with high aspect ratios (Type 3 substrates) during tugging at the pillars was investigated. Micropillars were produced using P(L-D,L)LA as well as a 60:40 blend of P(L-D,L)LA with PLGA. The blend is a material with lower stiffness than P(L-D,L)LA. The mechanical properties of the two materials were determined by tensile testing of solvent cast films. Deformation of Type 3 micropillars by the cellular tugging force of Saos-2 and L929 was studied by fluorescence and SEM microscopy, both on stiff and softer substrates. Displacements of the centers nodes of the pillars were evaluated from SEM micrographs. On the stiff surface, the two cell types bent the pillars to the same extent. On the other softer substrate (blends), however, the maximum displacements observed with Saos-2 cells were higher than the ones caused on the stiffer substrate or the ones caused by L929 cells. It is reported that stiffness of the substrate can determine stem cell lineage commitment. In order to examine the effects of change of substrate stiffness on osteogenic differentiation of BMSCs, osteopontin (OPN) expression was determined microscopically. It was found that osteogenic differentiation is enhanced when BMSCs are cultured on P(L-D,L)LA Type 3 pillars. vi In the last part of research, arrays of nanopillars whose interpillar distances systematically varied to form different fields were examined in terms of adhesion and alignment in order to determine the differential adhesion of BMSCs and Saos-2 cells. The difference in their adhesion preference on nanopillar arrays was quantified by image analysis. It was observed that BMSCs and Saos-2 cells behaved in an opposite manner with respect to each other on the fields with the highest density of nanopillars. The BMSCs avoided the most densely nanopillar covered fields and occupied the pattern free regions. The Saos-2, on the other hand, occupied the most densely nanopillar covered fields and left the pattern free regions almost unpopulated. It was also found that both BMSCs and Saos-2 cells aligned in the direction of the shorter distance between the pillars. Both BMSCs and Saos-2 cells started to align on the pillars if the distance in any direction was >1.5 μm. To better understand the effects of chemical and physical cues, protein coating and material stiffness were tested as two additional parameters. After fibronectin coating, the surfaces of P(L-D,L)LA films with the highly dense pillar covered fields, which were avoided when uncoated, were highly populated by the BMSC. Similarly, decreasing the stiffness of a surface which was normally avoided by the BMSCs made it more acceptable for the cells to attach.Ph.D. - Doctoral Progra

Üç farklı biyolaravasidal bakteri suşunda fermentasyon verimliliği analizi

The development of insecticides resistance among many insect species and the ecological damage occasionally caused by the lack of specificity in the toxic effects of insecticides have provided the impetus to seek alternative methods of insect control. This observation led to the development of bioinsecticides based on the insecticidal action Bacillus sphaericus (Bs), Bacillus turingiensis (Bt). The discovery of biolarvicidal actions of Bacillus thuringiensis and Bacillus sphaericus opened a new perspective for insect control. In the first part of the study was initiated to determine a suitable fermentation medium formulation and optimal fermentation conditions for large scale, low cost production of Bs. Bs 2362 was tested in whey and soy flour based media. These media was reformulized form of NYSM (Nutrient Broth Yeast Extract Sporulation Medium). Soy flour based medium, SYSM, gave the promising results in terms of cell yield, sporulation frequency and toxin production. In the second part of the study, fermentation productivity anlaysis of a local isolate Bacillus thuringiensis subsp. kurstaki 81 was evaluated. In order to compare different C:N ratios (1:1, 2:1, 4:1, 8:1, 10:1 20:1 and 30:1) of YSM medium. Btk 81 were run for 72 h and cell growth, sporulation and toxin protein profile of Btk 81 were determined for each. When all the quantitative toxin data for both glucose and sucrose varying C:N ratios were compared, it was determined that the crystal protein concentrations had the highest value in sucrose based medium when C:N ratio was 10:1. Regulation by C:N ratio of crystal protein biosynthesis was investigated for improving the production of this protein by our third candidate strain Bacillus thuringiensis subsp. israelensis ONR60. The experiments were performed by using TBL medium, at three different C:N ratios, 2:1, 4:1 and 8:1 respectively. In view of theM.S. - Master of Scienc

Trait anger and anger expression style in adolescents with chronic disease and determination of related factors

Kronik hastalıklar, ergenlerde birçok psikolojik ve duygusal problem neden olur. Akut hastalığa sahip ergenlerden farklı olarak, kronik hastalığa sahip bir ergenin çok sayıda ilaçlar, tedavi rejimleri, girişimler, diyetler gibi kronik ve sınırlayıcı koşullarda uzun süreler, hatta tüm hayatı boyunca yaşaması gerekir. Bu ergenlerde öfke önemli bir reaksiyondur. Bu çalışmanın amacı; kronik hastalığı olan ergenlerde sürekli öfke ve öfke ifade biçimlerini araştırmak ve akut hastalık nedeni ile hastaneye başvuran ergenler ile karşılaştırmak ve buna etki eden faktörleri araştırmaktır. Bu çalışmaya Hacettepe Üniversitesi Tıp Fakültesi, İhsan Doğramacı Çocuk Hastanesinde Nisan –Haziran 2014 tarihleri arasında izlenen yüz akut hastalığı olan ergen ve yüz kronik hastalığı olan ergen alınmıştır. Verileri toplamak için "Demografik Bilgi Formu" ve "Sürekli Öfke ve Öfke İfade Tarzı Ölçeği" kullanılmıştır. Akut ve kronik hastalık gruplarında sürekli öfke, içe yönelik öfke ve dışa yönelik öfke puanları arasında istatistiksel anlamlı fark izlenmemiştir. Fakat, kronik hastalık grubunda öfke kontrol puanları, akut hastalık grubundan anlamlı olarak yüksek bulunmuştur (p<0.011). Akut ve kronik hasta gruplarında; kızlar ve erkekler arasında ve yaş grupları arasında öfke ve öfke ifade puanları arasında fark bulunmamıştır. Ailenin büyüklüğü, kardeş sayısı ve ailenin ekonomik durumu gibi faktörlerin öfke ve ifadeleri üzerine bazı etkileri saptanmıştır. Kronik hastalığa sahip ergenler, akut hastalığa sahip ergenlere göre öfkelerini daha fazla kontrol edebilmektedirler.Chronic diseases cause many psychological and emotional problems in adolescents. Different from an adolescent with acute disease the adolescent with chronic disease must live in this chronic and restricted conditions with many drugs, treatment regimens, interventions, diets etc. for long periods even in his/her whole life. Anger is one of the important reaction in these adolescents. The aim of this study was to investigate trait anger and anger expression style of adolescents with chronic disease and compare with the adolescents admitted to the hospital for acute diseases and search for the related factors. A hundred adolescents with chronic diseases and a hundred adolescents with acute diseases, between the ages 12 to 18 years who were followed up in Hacettepe University Medical Faculty, İhsan Doğramacı Children's Hospital from April to June 2014 were included in this study. "Demographic Questionnaire" and "Trait Anger and Anger Expression Style Scale" were used to collect data. It was observed that there were not statistically important difference between acute and chronic disease groups for trait anger, and anger-in, anger-out sub-scales. But in chronic disease group, anger control scores were significantly higher than acute disease group (p<0.011). There were not differences between girls and boys, and age groups for anger and anger expression styles in acute and chronic disease groups. Family size, siblings number, and economical status of family have some effect on trait anger and anger expression styles. Adolescents with chronic disease were able to control their anger than the adolescents with acute disease

Nanodesenli hücre iskeleleri üretimi ve doku mühendisliği yöntemiyle yapay damar yapımında kullanımı

TÜBİTAK TBAG01.12.201

Ruthenium tris(2,2′-bipyridyl) complex encapsulated in nanosized faujasite zeolite as intracellular localization tracer

CERVOXYInternational audienceDesigning zeolites for medical applications is a challenging task that requires introducing new functionalities without altering the intrinsic properties such as morphology, crystallinity, colloidal stability, surface charge, and porosity. Herein, we present the encapsulation of luminescent ruthenium-tris(2,2′-bipyridyl) complex in faujasite (FAU) zeolite nanocrystals (Ru(bpy)3-FAU) and their use as an intracellular localization tracer. Upon exciting the Ru(bpy)3-FAU zeolite at 450 nm, the sample gives rise to an orange-red emission at 628 nm, thus permitting its use for cellular imaging and localization of the zeolite nanoparticles. The nanosized Ru(bpy)3-FAU zeolite is characterized in terms of size, charge, crystallinity, morphology, porosity, thermal stability, and sorption capacity. The potential toxicity of Ru(bpy)3-FAU on U251-MG glioblastoma cells was evaluated. A safe concentration (50-100 µg/ml) for the Ru(bpy)3-FAU zeolite is identified. The luminescent properties of the ruthenium complex confined in the zeolite nanocrystals allow their localization in the U251-MG cells with a main accumulation in the cytoplasm. The Ru(bpy)3-FAU nanosized zeolite is a potential candidate for biological applications for being stable, safe, capable of loading respiratory gases, and easily probed in the cells owing to its luminescent properties

Kinetics and oxygen dependence of uptake of nanozeolites by human glioblastoma cells

CERVOXYNational audienc

Internalization study of nanosized zeolite crystals in human glioblastoma cells

CERVOXYInternational audienceWhile the use of nanozeolites for cancer treatment holds a great promise, it also requires a better understanding of the interaction between the zeolite nanoparticles and cancer cells and notably their internalization and biodistribution. It is particularly important in situation of hypoxia, a very common situations in aggressive cancers, which may change the energetic processes required for cellular uptake. Herein, we studied, in vitro, the kinetics of the internalization process and the intracellular localization of nanosized zeolite X (FAU-X) into glioblastoma cells. In normoxic conditions, scanning electron microscopy (SEM) showed a rapid cell membrane adhesion of zeolite nanoparticles (< 5 min following application in the cell medium), occurring before an energy-dependent uptake which appeared between 1 h and 4 h. Additionally, transmission electron microscopy (TEM) and flow cytometry analyzes, confirmed that the zeolite nanoparticles accumulate over time into the cytoplasm and were mostly located into vesicles visible at least up to 6 days. Interestingly, the uptake of zeolite nanoparticles was found to be dependent on oxygen concentration, i.e. an increase in internalization in severe hypoxia (0.2 % of O2) was observed. No toxicity of zeolite FAU-X nanoparticles was detected after 24 h and 72 h. The results clearly showed that the nanosized zeolites crystals were rapidly internalized via energy-requiring mechanism by cancer cells and even more in the hypoxic conditions. Once the zeolite nanoparticles were internalized into cells, they appeared to be safe and stable and therefore, they are envisioned to be used as carrier of various compounds to target cancer cells