Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    GO-FRESH: Valorisatie kansrijke oplossingen voor een robuuste zoetwatervoorziening : Rendabel en duurzaam watergebruik in een zilte omgeving
    Veraart, J.A. ; Oude Essink, G. ; Pauw, P. ; Baaren, E. van; Zuurbier, K. ; Louw, P. de; MacAteer, E. ; Schoot, M. van der; Groot, N. ; Cappon, H. ; Waterloo, M. ; Hu-a-ng, K. ; Groen, M. - \ 2018
    Deltares - 187 p.
    An overview of microplastic and nanoplastic pollution in agroecosystems
    Ng, Ee Ling ; Huerta Lwanga, Esperanza ; Eldridge, Simon M. ; Johnston, Priscilla ; Hu, Hang Wei ; Geissen, Violette ; Chen, Deli - \ 2018
    Science of the Total Environment 627 (2018). - ISSN 0048-9697 - p. 1377 - 1388.
    Ecotoxicology - Plant response - Plastic degradation - Soil food web - Soils
    Microplastics and nanoplastics are emerging pollutants of global importance. They are small enough to be ingested by a wide range of organisms and at nano-scale, they may cross some biological barriers. However, our understanding of their ecological impact on the terrestrial environment is limited. Plastic particle loading in agroecosystems could be high due to inputs of some recycled organic waste and plastic film mulching, so it is vital that we develop a greater understanding of any potentially harmful or adverse impacts of these pollutants to agroecosystems. In this article, we discuss the sources of plastic particles in agroecosystems, the mechanisms, constraints and dynamic behaviour of plastic during aging on land, and explore the responses of soil organisms and plants at different levels of biological organisation to plastic particles of micro and nano-scale. Based on limited evidence at this point and understanding that the lack of evidence of ecological impact from microplastic and nanoplastic in agroecosystems does not equate to the evidence of absence, we propose considerations for addressing the gaps in knowledge so that we can adequately safeguard world food supply.
    Relationships between uterine health and metabolism in dairy cows with different dry period lengths
    Chen, J. ; Soede, N.M. ; Remmelink, G.J. ; Bruckmaier, R.M. ; Kemp, B. ; Knegsel, A.T.M. van - \ 2017
    Theriogenology 101 (2017). - ISSN 0093-691X - p. 8 - 14.
    Continuous milking - Glucogenic nutrient - Lipogenic nutrient - Progesterone - Uterine health

    The first objective of this study was to evaluate effects of dry period (DP) length and dietary energy source on ovarian activity, uterine health status, pregnancy rate, and days open in dairy cows in the second subsequent lactation after implementation of DP length and dietary treatments. The second objective was to determine relationships of uterine health status with ovarian activity, milk yield, energy balance (EB), and metabolic status in dairy cows. Holstein-Friesian dairy cows (n = 167) were assigned randomly to 1 of 3 DP lengths (0-, 30-, or 60-d) and 1 of 2 early lactation diets (glucogenic or lipogenic diet) for 2 subsequent lactations. Milk samples were collected three times a week. At least two succeeding milk samples with concentration of progesterone ≥2 ng/mL were used to indicate the occurrence of luteal activity. Vaginal discharge was scored in wk 2 and 3 after calving to evaluate uterine health status and cows were classified as having a healthy uterine environment [HU, vaginal discharge score (VDS) = 0 or 1 in both wk 2 and 3], a recovering uterine environment (RU, VDS = 2 or 3 in wk 2 and VDS = 0 or 1 in wk 3), or a non-recovering uterine environment (NRU, VDS = 2 or 3 in wk 3). Cows were monitored for milk yield, dry matter intake (DMI), and blood was sampled weekly to determine metabolic status from calving to wk 3 postcalving. Dry period length was not related with uterine health status in early lactation, pregnancy rate, or days open in dairy cows. Independent of DP length, feeding a glucogenic diet shortened the interval from calving to onset of luteal activity (25.3 vs. 31.0 d, P = 0.04), but decreased pregnancy rate compared with a more lipogenic diet (68.2 vs. 78.1 d, P = 0.03). In the first 3 wk after calving, cows with a NRU had lower milk yield (36.8 vs. 36.8 vs. 32.4 kg for cows with a HU, RU, or NRU, respectively; P < 0.01) and lower DMI than cows with a HU or RU. Cows with a RU had lower plasma glucose and insulin concentrations than cows with a NRU or HU. In conclusion, DP length did not influence fertility measures and uterine health status in the second subsequent lactation after implementation of DP length treatments. Independent of DP length, feeding a glucogenic diet leaded to earlier ovulation postcalving, but decreased pregnancy rate compared with a more lipogenic diet. In addition, a healthy uterine environment was related to greater milk yield and better metabolic status, independent of DP length.

    Subgenome parallel selection is associated with morphotype diversification and convergent crop domestication in Brassica rapa and Brassica oleracea
    Cheng, Feng ; Sun, Rifei ; Hou, Xilin ; Zheng, Hongkun ; Zhang, Fenglan ; Zhang, Yangyong ; Liu, Bo ; Liang, Jianli ; Zhuang, Mu ; Liu, Yunxia ; Liu, Dongyuan ; Wang, Xiaobo ; Li, Pingxia ; Liu, Yumei ; Lin, Ke ; Bucher, Johan ; Zhang, Ningwen ; Wang, Yan ; Wang, Hui ; Deng, Jie ; Liao, Yongcui ; Wei, Keyun ; Zhang, Xueming ; Fu, Lixia ; Hu, Yunyan ; Liu, Jisheng ; Cai, Chengcheng ; Zhang, Shujiang ; Zhang, Shifan ; Li, Fei ; Zhang, Hui ; Zhang, Jifang ; Guo, Ning ; Liu, Zhiyuan ; Liu, Jin ; Sun, Chao ; Ma, Yuan ; Zhang, Haijiao ; Cui, Yang ; Freeling, Micheal R. ; Borm, Theo ; Bonnema, Guusje ; Wu, Jian ; Wang, Xiaowu - \ 2016
    Nature Genetics 48 (2016)10. - ISSN 1061-4036 - p. 1218 - 1224.

    Brassica species, including crops such as cabbage, turnip and oilseed, display enormous phenotypic variation. Brassica genomes have all undergone a whole-genome triplication (WGT) event with unknown effects on phenotype diversification. We resequenced 199 Brassica rapa and 119 Brassica oleracea accessions representing various morphotypes and identified signals of selection at the mesohexaploid subgenome level. For cabbage morphotypes with their typical leaf-heading trait, we identified four subgenome loci that show signs of parallel selection among subgenomes within B. rapa, as well as four such loci within B. oleracea. Fifteen subgenome loci are under selection and are shared by these two species. We also detected strong subgenome parallel selection linked to the domestication of the tuberous morphotypes, turnip (B. rapa) and kohlrabi (B. oleracea). Overall, we demonstrated that the mesohexaploidization of the two Brassica genomes contributed to their diversification into heading and tuber-forming morphotypes through convergent subgenome parallel selection of paralogous genes.

    Evaluation of Harmonic Analysis of Time Series (HANTS): impact of gaps on time series reconstruction
    Zhou, J.Y. ; Jia, L. ; Hu, G. ; Menenti, M. - \ 2012
    In: Proceedings of the Second International Workshop on Earth Observation and Remote Sensing Applications (EORSA 2012), Shanghai, China, 8-11 June 2012. - Shanghai, China : IEEE Xplore - ISBN 9781467319478 - p. 31 - 35.
    In recent decades, researchers have developed methods and models to reconstruct time series of irregularly spaced observations from satellite remote sensing, among which the widely used Harmonic Analysis of Time Series (HANTS) method. Many studies based on time series reconstructed with HANTS documented the excellent performance of this method. While some limitations of HANTS have been noticed in these applications, there is no dedicated study on a systematic evaluation on the performance of the HANTS method. In this study, we evaluated the impact of gaps on the time series reconstruction of NDVI by HANTS. For global representativeness, a simulated NDVI time series dataset was constructed for four generic patterns and was applied as a reference dataset. Then random gaps were introduced into the reference series and both the reference and gapped series were reconstructed by harmonic analysis. The deviations between the two reconstructed results were used to evaluate statistically the accuracy of harmonic analysis under different gap conditions. The size of maximum gap (MGS), the number of loss (NL) and the number of gaps (NG) were selected to parameterize the gap distribution. The results showed that MGS, NL and NG were significant factors in the process of reconstruction and the two terminals and the peak of the series are crucial positions. MGS and NL should not be too large in the time series for all seasonal or non-seasonal case; otherwise the reconstructed series is not reliable. These conclusions can be taken as a reference to indicate the reliability of HANTS for particular cases towards the definition of a quality indicator of any time series.
    Collagen-inspired self-assembling materals
    Skrzeszewska, P.J. - \ 2011
    Wageningen University. Promotor(en): Martien Cohen Stuart, co-promotor(en): Jasper van der Gucht; Frits de Wolf. - [S.l.] : S.n. - ISBN 9789085858690 - 151
    zelf-assemblage - polypeptiden - biodegradatie - pichia pastoris - genetische modificatie - genetisch gemanipuleerde micro-organismen - aminozuursequenties - biomedische techniek - self assembly - polypeptides - biodegradation - pichia pastoris - genetic engineering - genetically engineered microorganisms - amino acid sequences - biomedical engineering

    The rapid increase of the quality of life together with the progress of medical science asks for the development of new, tuneable and controllable materials. For the same reason, materials used for biomedical applications have to be increasingly biocompatible, biodegradable and biofunctional. Most of the available systems, however, lack one property or the other. For example, conventional animal-derived gelatin that is often used in biomedicine, is susceptible to a risk of contamination with prions or viruses and has a risk of bringing out allergic reactions, particularly against the non helix-forming domains of collagen [1]. Furthermore, gelatin is composed of a variety of molecules and structures with different thermal stabilities and molecular sizes. This, in combination with the impossibility to change the molecular structure at will, limits the chances to elucidate the relation between the structure and function. On the other hand, synthetic materials that have a rather well-controlled size distribution often lack biocompatibility, biofunctionality or biodegradability. In addition to that, as their synthesis often requires toxic solvents, their application in the human body is restricted. All the drawbacks of the presently used materials have brought scientists towards a new approach in designing materials viz. genetic engineering. Rapid progress in recombinant techniques has led to new ways of producing molecules with well-defined composition and structure and with full control over the length and sequence of the biopolymer and its constituent blocks. These methods thus combine the advantages of natural and synthetic polymers. Using molecular biology tools, unique molecules can be created by merging in a desired manner naturally occurring self-assembling motifs such as elastin, silk or collagen [2-4], or entirely artificial fragments. As we show in this thesis, the precise control over the molecular design of these biotechnologically produced block polypeptides is extremely valuable as it also leads to control over their physicochemical properties.

    In this thesis we present a new class of monodisperse, biodegradable and biocompatible network-forming block polymers that are produced by genetically modified strain of yeast, Pichia pastoris (Chapter 2). Trimer-forming end blocks, abbreviated as T, consisting of nine Pro-Gly-Pro amino acid triplets, are symmetrically flanking a random coil-like middle block composed of four or eight repeats of highly hydrophilic R or P sequences (Figure 8.1). R and P are identical with respect to length (99 amino acids) and composition but have different amino acid sequences. The P block has a glycine in every third position (as in collagen) but does not form any supramolecular structures and maintains a random coil-like conformation at any temperature [5]. The R block is a shuffled version of the P block. Four recombinant gelatins are reported in this thesis, denoted as TR4T, TR8T, TP4T and TP8T (Figure 8.1). All of these were successfully produced with high yields (1-3 g/l of fermentation broth) by the Pichia pastoris GS115 strain transformed with a pPIC9 vector with the gene of interest in its expression cassette.

    Figure 8.1 Schematic representation of collagen-inspired telechelic polypeptides: TR4T, TR8T, TP4T and TP8T.

    In Chapter 3 we described the linear rheological properties of hydrogels formed by TR4T polypeptides. At a temperature of 50 °C, the solution does not show any viscoelastic response. However, upon cooling, the collagen-like trimer-forming domains (T) start to assemble into triple helical nodes and a well-defined network, with a node multiplicity of three, is formed. In the beginning of the gelation process, viscous properties are predominant, but as the network formation progresses, the elastic properties prevail. A plateau storage modulus is reached within a few hours. At this point the triple helices are in equilibrium with the free T blocks. An equilibrium or near-equilibrium state is reached, contrary to natural gelatin, because the collagen-like (T) assembling domains are relatively short and well-defined. The T blocks are solely responsible for the network formation. We have shown that a solution of the middle blocks only (i.e. R4) does not demonstrate any elastic response at any time and temperature. In addition, differential scanning calorimetry (DSC) (Chapter 5) proved that the collagen-like side blocks are near-quantitatively responsible for trimerization, as the observed melting enthalpies are in good agreement with values obtained by Frank et al. [6] for free (Pro-Gly-Pro)10 peptides. The equilibrium fraction of T blocks involved in triple helices shifts with temperature. By lowering the temperature, the fraction of triple helices increases, while the fraction of free ends decreases. There are two possibilities to form a triple helix. It can be formed either by three T blocks from three different chains, or by three T blocks from two different chains, so that two side blocks come from the same polypeptide. As a consequence, the network is composed of dangling ends, elastically active bridges and inactive loops (Figure 8.2). Because of the precisely-known junction multiplicity of three, we could develop an analytical model that links the internal structure of the gel, with dangling ends, loops, and bridges, to the physicochemical properties. This model uses a limited set of input parameters that can all be measured independently. It describes the experimental data quantitatively without further adjustable parameters. Using this model, we could show that the observed strong dependency of the storage modulus, the relaxation time and the viscosity on concentration and temperature is related to the changes in the number of loops, active bridges, and dangling ends in the gel matrix.

    Figure 8.2 Network formation by collagen-inspired telechelic biopolymers.

    In Chapter 4 we show that the number of intermolecular junctions and intramolecular loops depends not only on protein concentration and temperature but also on the length and the stiffness of the middle block. We synthesised new triblock copolymers with middle blocks, of different lengths and amino acid sequences, named TP4T, TR8T and TP8T (Figure 8.1). For all new proteins, there is a strong dependency of the storage modulus, the relaxation time and the viscosity on concentration and temperature (as for TR4T). However at comparable molar concentrations, the longer versions of polypeptides i.e. TR8T and TP8T show a significantly higher storage modulus and relaxation time than their counterparts TR4T and TP4T. This is because a longer middle block leads to a larger radius of gyration (Rg), which decreases the probability that two end blocks from the same molecule associate with each other, and form a loop. The consequence of fewer loops in the system is a higher storage modulus and a higher overall relaxation time.



    Besides the effect of polymer length, we also observed that the R series, i.e. TR4T and TR8T, show a higher storage modulus than their P counterparts, i.e. TP4T and TP8T, at the same concentration and temperature. This can be explained by differences in coil flexibility. Although the P and R blocks have exactly the same amino acid composition, their amino acid sequence is different. Fitzkee et al. [7] have shown that even a polypeptide chain that assumes a random coil conformation still has locally folded conformations that contribute to the overall flexibility of the chain. This apparently leads to a smaller radius of gyration for the P middle block than for the R middle block and thus to a higher probability of loop formation.

    Even though the melting behaviour obtained with DSC is the same for all four polypeptides (as the end blocks stay the same), the temperature at which the G0 value approaches zero and the gel completely loses its elastic properties varies with the length of the middle block. Shorter molecules, i.e. TP4T and TR4T, melt at lower temperatures. A solution of 1.2 mM TP4T melts at 298 K, while TP8T at a comparable molar concentration melts at a temperature which is 15 degrees higher. Furthermore, the R versions show slightly higher melting temperatures than the P versions. These differences in melting behaviour are related to the gel structure and the relative probabilities of forming intramolecular and intermolecular assemblies. We could account for these findings with the help of the analytical model presented in Chapter 3. The only parameter that had to be varied in the model was the coil size of the polymer, since the enthalpy and the melting temperatures of the triple helices did not change with the length of the middle block. The theoretical calculations clearly show that the molecules with smaller Rg form up to 30 % more loops than their bigger counterparts. Loops that act as gel stoppers do not contribute to the network elasticity and significantly lower the melting temperatures detected with rheology.

    The network junctions in our gels are solely formed by triple helices. The mechanism of junction formation by the T blocks can be well-described by a two-step kinetic model (Chapter 5). Prior to triple helix propagation, a trimeric nucleus has to be formed. For dilute systems, nucleation is the limiting step, giving an apparent reaction order of three. These results indicate that only triple helices are stable. For more concentrated solutions, when nucleation is relatively fast, propagation of triple helices becomes rate-limiting and the apparent reaction order is close to unity. The propagation of triple helices is probably limited by cis-trans isomerization of peptide bonds, in which proline residues are involved.

    Above overlap concentration (C*) the measured enthalpy for stable gels (~15 hours) indicates that almost 100 % of the T blocks are involved in triple helices. Values obtained by us are in good agreement with values obtained by Frank et al. [6] for single (Gly-Pro-Gly)10 peptides. Conversely, at concentrations below C*, the enthalpy per mole of protein is becoming less, suggesting that the fraction of free ends or mismatched helices becomes more pronounced. The apparent melting temperature increases slightly with increasing concentration. This can be explained on the basis of the reaction stoichiometry under equilibrium conditions [8, 9]. Except for the highest measured concentration (2.4 mM), the apparent melting temperature revealed a dependence on the scan rate, indicating that it was not possible to maintain equilibrium during the heating step. At a concentration of 2.4 mM concentration there is no scan rate dependence, since the melting occurs at a higher temperature, where the dissociation kinetics is faster [4, 10].

    The kinetics of triple helix formation determines the rate of gel formation. The gelation starts when the first triple helical node is formed. At that time viscous properties (loss modulus) predominate, but as the network formation evolves the elastic response (storage modulus) becomes more pronounced. The storage modulus (G’) reaches a plateau value within a few hours. Changes in network structure and mechanical properties of the gel in time can be predicted from the kinetics of triple helix formation, using the model presented in Chapter 3. By comparing the kinetics obtained with rheology and with DSC we could see that for our system, the helix content is not simply proportional to the network progress and that the relation between the elastic properties (G’) and the helix content (pH) depends on the protein concentration. The reason for this concentration dependence is the formation of loops, which is more likely at low concentrations.

    The investigated hydrogels undergo time-dependent macroscopic fracturing when a constant shear rate or shear stress is applied (start-up and creep experiments, respectively) (Chapter 6). Observations with particle image velocimetry (PIV) showed that in the beginning of a start-up (or creep) experiment the sample flows homogenously. After some time, the gel fractures, and is separated into two fractions. The inner region moves at the same velocity as the moving bob, while the outer fraction does not move at all. From the rate-dependence of the fracture strength we can conclude that gel fracture is due to stress-activated rupture of the triple helical nodes in the network.When the deformation is taken away, the gel can heal (Chapter 6). The capacity of self-healing is due to the transient character of the network nodes with a finite relaxation time. Such behaviour, impossible for most permanent gels, is highly desired in many applications, as hydrogels are often subjected to deformations, which easily go beyond the linear regime. As we present in Figure 8.3, TR4T gels cut into small pieces (grey and transparent), can heal within 2 hours. As measured with rheology the broken gel can recover up to 100 % of its initial elastic properties, even after several fracturing cycles. Interestingly, the kinetics of healing differs from the kinetics of fresh gel formation (Chapter 5). The latter is characterized by a lag-phase before elastic properties start to appear. This lag-phase occurs because at low degrees of crosslinking there is not yet a percolated network, so that the storage modulus is undetectable. By contrast, the recovery of the gel after rupturing is much faster and does not show a lag-phase. The elastic modulus, depending on the rupturing history, comes back to its initial value within 1-5 hours. These findings indicate that outside the fracture zone, the network nodes have not dissociated significantly, so that healing only requires the reformation of junctions that connect the undamaged pieces of the network (gel clusters).

    Figure 8.3 Self-healing of TR4T hydrogels. (A) Pieces of broken gel. (B) Two gel pieces healed after 2 hours.

    In Chapter 7 we demonstrated the shape-memory effects in hydrogels formed by permanently crosslinked TR4T molecules. The programmed shape of these hydrogels was achieved by chemical crosslinking of lysine residues present in the random coil. The chemical network could be stretched up to 200 % and “pinned” in a temporary shape by lowering the temperature and allowing the collagen-like end blocks to assemble into the physical nodes. The deformed shape of hydrogel can be maintained, at room temperature, for several days, or relaxed within few minutes upon heating to 50 ºC or higher. The presented hydrogels could return to their programmed shape even after several thermo-mechanical cycles, hence indicating that they remember the programmed shape. We have studied in more detail the shape recovery process by describing our hydrogels by a mechanical model composed of two springs and a dashpot. With the help of this model we showed that above the melting temperature of the triple helices, the recovery is exponential and that the decay time is roughly ten times slower than the relaxation of the physical network.

    1.Biomedical applications - perspectives and considerations

    The class of collagen-inspired self-assembling materials, which we present in this thesis are nice model systems for a systematic study of physical networks, but they also have a lot of potential for biomedical applications. In this section we discuss the possibilities for these self- assembling hydrogels in biomedicine.

    Drug delivery systems

    One of the major goals of modern medicine is to ensure that the required amount of an active substance is available at the desired time at the desired location in the body. Consequently, a lot of effort is put into designing delivery systems with precisely adapted release profiles, sensitive to external stimuli such as temperature or pH. A frequently used group of materials in this field are hydrogels, both chemically and physically crosslinked. In the case of covalently crosslinked networks the release of the drug is mostly via diffusion of the drug out of the gel particle after it swells. The rate of drug release is governed by the resistance of the network to volume increase [11]. Although permanent networks are widely used as drug carriers they have some disadvantages such as incomplete release of active substances and poor biodegradability in the body. The problem can be partially solved by introducing enzymatic cleavage or hydrolysis sites into the main chain, but still the hydrogel erosion cannot be precisely controlled and complete material degradation can not be guaranteed.

    These obstacles can be overcome by using physical hydrogels that are formed by weak interactions. These can dissociate in a controlled manner and completely release the active component. In contrast to chemical gels, erosion of physical gels occurs spontaneously. The erosion rate is determined by the life time of the junctions, but it depends also on the relative amount of intramolecular loops and intermolecular junctions, as demonstrated by Shen et al. [12]. These authors showed that, by using triblock polymers with dissimilar coiled-coil side domains rather than identical ones, loop formation could be suppressed, leading to a lower erosion rate [12].

    The potential of our gels for drug delivery applications was tested by Teles et al. [13]. It was shown that trapped proteins (BSA) can be completely released from TR4T and TR8T gels, both at 37 ºC and 20 ºC. The release at 37 ºC from 20 % gels was completed within 48 hours while at 20 ºC it took about 5 times longer. At body temperature the release was mostly driven by dissociation of trimeric junction and dissolution of the separate polymer chains (gel erosion). At 20 ºC the junction life time was long so that erosion was slower and swelling and diffusion played a more important role. The observations of Teles are in agreement with studies of several groups that demonstrated the importance of hydrogel erosion for controlled release [12, 14].

    The erosion rate of physical hydrogels is governed by the junction relaxation time. The mean relaxation time of transient networks can be manipulated either by varying the gel architecture (Chapter 3 and 4) or by changing the relaxation time of a single triple helix. The gel architecture (i.e. the number of loops and bridges) can be altered either as we show in Chapter 3 by changing the protein concentration or as we demonstrate in Chapter 4 by manipulating the design of the middle block. The number of loops becomes lower as the spacer length and stiffness increase (Chapter 4). The lifetime of a single node can be changed by enzymatic hydroxylation of proline to hydroxyproline, [15] which leads to more hydrogen bonds among adjacent T blocks, or by changing the length of the collagen-like T domains. Preliminary results showed that average relaxation time of the network is roughly hounded times higher for molecules with collagen-like domains composed of sixteen Pro-Gly-Pro repeats instead of nine (unpublished data).

    For these biotechnologically produced collagen-inspired polymers, the length or the composition of the blocks can be changed simply by changing the DNA template. This, in combination with the model elaborated in Chapter 3 and 4 that links the internal gel architecture with the physicochemical gel behaviour, gives ample possibilities to design materials with custom-desired release profiles of active components.

    Tissue engineering

    Materials for tissue engineering scaffolds have to mimic the in vivo extracellular matrix environment. They provide physical support, but also have to guarantee proper adhesion of cells and controlled release of growth factors. A very important role in scaffolds design is played by the mechanical properties of the matrix [16-18]. As shown by Engler et al. [17], the elasticity of the matrix directs stem cell development to different lineages. Soft networks (0.1-1 kPa), which mimic brain tissue, promote neuron development, stiffer scaffolds (8-17 kPa) are myogenic, while gels with an elastic modulus of 24-40 kPa promote growth of bone cells. The stiffness of the matrix affects focal-adhesions and the organization of the cytoskeleton structure, and thus contractility, motility and spreading [16, 18]. Another significant factor, which plays a role in tissue growth is the degradation rate of the scaffold. The degradation should be synchronized with cellular repair in such a way, that tissue replaces the material within the desired time interval. The scaffold disintegration also controls the release of growth factors. For naturally derived materials such as alginate, the degradation rate could be influenced by partial oxidation of the polymer chain or via a bimodal molecular weight distribution [19]. For synthetic polymers different degradation profiles can be realized by incorporating in the polymer backbone groups with different susceptibility to hydrolysis [19].

    Presently the most widely used scaffolds for tissue engineering are natural polymers such as collagen, gelatin, and polysaccharides [20] or synthetic, biodegradable polymers such as poly (L-lactic acid) (PLLA), poly(glycolic acid) (PGA), and poly(ethylene glycol) (PEG). [21-23]. Although these materials show promising properties, their use is limited as they suffer from batch to batch variations, polydispersity, viral contamination, allergic reactions or toxic byproducts after degradation. Also their mechanical properties are poorly-controlled and it is difficult to relate the molecular structure to the resulting properties. Furthermore, in the case of synthetic polymers, there is no intrinsic mechanism to interact with cells and to propagate cell adhesion proliferation or migration. This problem can be partially solved by functionalizing synthetic materials with bioactive molecules, such as collagen [24] or short peptides (for example arginine-glycine-aspartic (RGD) or tyrosine-isoleucine-glycine-serine-arginine (YIGSR) [25]). It remains difficult, however, to precisely control the spatial distribution, of these biofunctional domains [25].

    A very promising alternative for the currently used scaffolds are hydrogels formed by self-assembling protein polymers [2, 26-28], including the collagen-inspired polypeptides presented in this thesis. Our block polymers form physical gels with precisely controlled elastic properties. As discussed in Chapter 3 and 4, the gel structure and the resulting mechanical properties strongly depend on concentration, temperature and on the molecular design of the polymer. Within the investigated range of conditions our gels have an elastic modulus between 0.03 and 5 kPa. Thus they seem most appropriate for neuron cell growth [17]. Moreover, it is also possible to incorporate specific short adhesive peptide sequences (such as RGD) in the middle block to improve attachment and cell propagation.

    The presently investigated proteins, with T domains composed of nine Pro-Gly-Pro repeats, still need some enhancement in terms of stability. As shown by Teles et al. the currently available molecules erode within 2 days [13]. For tissue engineering applications, this is too fast. We therefore propose some strategies to stabilize our hydrogels. A first possibility alternative is to introduce amino acids which can form chemical bonds such as cysteines that can form disulfide bridges under oxidizing conditions [29], or lysines, which can be functionalized with acrylate and then photo-crosslinked with UV radiation [30-32]. However, one has to be aware that this additional procedure may have negative side effects such as toxic byproducts, incomplete polymer degradation in the body, or loss of responsiveness to external stimuli. Alternatively, the erosion can be moderately slowed down by increasing the relaxation time of the network (as discussed in section on drug delivery systems).

    Wound dressing materials

    Under normal circumstances wound healing is a very long process. In order to speed it up, so that bacterial infections or wound dehydration can be avoided, wound dressing materials are used [33-37]. These materials should fulfil several general requirements such as biocompability, ease of application and removal, proper adherence (to avoid fluid pockets, in which bacteria could proliferate), ease in gas exchange between tissue and environment, and controlled release of active components such as antimicrobial agents or wound repair agents (for example Epidermal Growth Factor (EGF)) [37].

    All above-mentioned requirements can be fulfilled by the collagen-inspired hydrogels presented in this thesis. The advantage of our materials is that they can follow the contour of the wound and entirely fill it, thus forming an efficient barrier for microbes, but at the same time being permeable for water vapour and oxygen. Furthermore they can entrap active components and release them in a controlled way during the healing process, as discussed above. Depending on the circumstances, the release profile can be synchronised with the wound healing process. An additional advantage of our genetically engineered molecules is that adhesion domains can be introduced along the middle block, assuring better integration of the gel with the damaged tissue.

    8.3 Final conclusions and outlook

    In this final chapter we have discussed the potential of our collagen-inspired materials in biomedical applications. They are biocompatible and biodegradable, whilst offering numerous possibilities to change the molecular design in order to meet the desired mechanical or biological properties. Furthermore, the well-defined nature of the triple helical junctions allows us to predict the mechanical properties of the gel from the molecular design of polypeptides. This exclusive feature of our system makes it unique and offers great flexibility to design custom biomedical materials.

    Biomedical needs, however, are very variable and often require an individual approach. That is why in our group we have created a family of genetically engineered block copolypeptides. Besides collagen we use other motifs present in nature, such as silk or elastin. We can combine these motifs in various ways in order to create unique stimuli-responsive (often multi-responsive) molecules that can meet individual application needs.

    The silk-like domains consist of (Gly-Ala-Gly-Ala-Gly-Ala-Gly-Xxx)n repeats. Position Xxx is occupied by charged amino acids such as histidine, lysine or glutamic acid. When the charge is screened, the molecules assemble, forming first β sheet-like secondary structures, and then long fibres. As shown by Martens et al. [38], block polymers comprising silk-like domains with glutamic acid or histidine in the Xxx position form fibre-like gels at a pH of 2 or 12, respectively. They also assemble when mixed with oppositely charged (coordination) polymers [3, 38]. Probably, the assembling conditions can be tuned even more precisely by adjusting the isoelectric point of the assembling domain. This will allow the production of hydrogels that are formed after being injected into the body, while they disassemble (releasing the drug) when exposed to the acidic or the alkaline conditions. The nanofibre gels are also stable enough to serve as scaffolds for tissue engineering [39-41].

    Another motif that has been used is elastin. It consists of (Val-Pro-Gly-Xxx-Gly)n repeats and it self-assembles above a lower critical solution temperature (LCST). The transition temperature can be tuned by introducing more or less polar amino acid residues in position Xxx. By combining elastin-like or collagen-like blocks with silk-like blocks, thermo and pH responsive networks can be obtained. This may allow us toswitch from fibre-like gels to associative networks.

    Block polypeptides, produced using recombinant techniques, besides biocompability and biodegradability offer many possibilities to adjust the molecular design in will, to realize the desired mechanical or biological properties. Three dimensional structures with different thermal stabilities can be programmed by combining in a precise manner various amino acid sequences. The obtained materials can respond to external stimuli such as pH, ionic strength or temperature. They can also carry peptides fragments that can enhance cells adhesion and proliferation or induce crystallization.

    The new approach in material science, which we present in this thesis, opens a new world of polymers, in which the main constraint is imagination.


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    Polycyclic aromatic hydrocarbons in soils around Guanting Reservoir, Beijing, China
    Jiao, W.T. ; Lu, Y.L. ; Wang, T.Y. ; Li, J. ; Han, Jingyi ; Wang, G. ; Hu, W.Y. - \ 2009
    Chemistry and Ecology 25 (2009)1. - ISSN 0275-7540 - p. 39 - 48.
    pahs - contamination - sediments - tianjin - urban
    The concentrations of 16 polycyclic aromatic hydrocarbons ( 16PAHs) were measured by gas chromatography equipped with a mass spectrometry detector (GC-MS) in 56 topsoil samples around Guanting Reservior (GTR), which is an important water source for Beijing. Low to medium levels of PAH contamination (mean=394.2580.7ngg-1 dry weight (d.w.)) was evident throughout the region. In addition, localised areas of high PAH contamination near steel and cement factories were identified, with 16PAHs concentrations as high as 4110ng/g, dry weight (d.w.). There was a significant positive correlation (r2=0.570, p0.01) between total organic carbon content and 16PAHs concentrations. Phenanthrene was the predominant compound, accounting for 27.2% of the PAH concentration, followed by chrysenepyrenebenzo[a]anthracene benzo[b]fluoranthene benzo[a]pyrene. Four-ring PAH homologues (39%) were dominant. The higher proportion of 4-6 ring homologues, molecular indices, and the spatial distribution of PAH indicated that industrial discharges, incineration of wastes and traffic discharges were the major sources of soil PAHs around the water reservoir.
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