Cell-matrix interaction in tissue patterning

Abstract only availableIn vivo pattern formation during morphogenesis is dependent upon the migration of cells. Cell movements are directed by the local structure of the surrounding extracellular matrix. It has been shown experimentally that ligament-like straps form in collagen gel due to the local tension created in the matrix by tissue explants [1]. For a comprehensive understanding of this phenomenon, the sprouting behavior of these aggregates was qualitatively studied in six different configurations embedded in a collagen gel. The biological motivation for this study was to observe how the interplay between the collagen matrix, simulating the extracellular matrix, and the cells affect pattern formation in tissues. The understanding of tissue patterning as a result of cell-matrix interaction has important implications for tissue engineering. Spherical aggregates were prepared from Chinese Hamster Ovary Cells as described previously [2]. The six configurations (triangular, square, hexagonal, bulls eye, dodecagon, and two adjacent aggregates) were built by manually placing spherical aggregates into collagen gels. Photographs of the evolving patterns were taken at regular time intervals for one hundred and eighty hours under a phase contrast microscope. Sprouting was delayed until a critical tension was reached in the collagen matrix. Once sprouting began, a clear bias was shown for migration of cells toward other aggregates creating a cellular bridge between aggregates in close proximity. Sprouting occurred toward each aggregate in a specific pattern exhibiting anisotropy due to the depletion of local collagen fibers in areas adjacent to the cellular bridges. In most aggregates, a void in cell sprouting was apparent on either side of the cellular bridge. The large-scale patterns exhibited in this experiment were found to be linked to local cell-matrix interactions. [1] Sawhney, R.K, Howard, J, Slow local movements of collagen fibers by fibroblasts drive the rapid global self-organization of collagen gels. Journal of Cell Biology. Vol 157, 6, 2002, pp. 1083-1091. [2] K. Jakab, A. Neagu, V. Mironov, R.R. Markwald and G. Forgacs. Engineering biological structures of prescribed shape using self-assembling multicellular systems. PNAS, vol. 101, 9, pp. 2864-2869, 2004.NSF-REU Biosystems Modelin

Role of Physical Mechanisms in Biological Self-Organization

URL:http://link.aps.org/doi/10.1103/PhysRevLett.95.178104 DOI:10.1103/PhysRevLett.95.178104Organs form during morphogenesis, the process that gives rise to specialized biological structures of specific shape and function in early embryonic development. Morphogenesis is under strict genetic control, but shape evolution itself is a physical process. Here we report the results of experimental and modeling biophysical studies on in vitro biological structure formation. Experimentally, by controlling the interaction between cells and their embedding matrices, we were able to build living structures of definite geometry. The experimentally observed shape evolution was reproduced by Monte Carlo simulations, which also shed light on the biophysical basis of the process. Our work suggests a novel way to engineer biological structures of controlled shape.This work was supported by NSF (IBN-0083653; FIBR-0526854) and NASA (NAG2-1611)

Interplay between JA, SA and ABA isgnalling during basal and induced resistance against pseudomonas syringae and Alternaria brasicicola

We have examined the role of the callose synthase PMR4 in basal resistance and β‐aminobutyric acid‐induced resistance (BABA‐IR) of Arabidopsis thaliana against the hemi‐biotrophic pathogen Pseudomonas syringae and the necrotrophic pathogen Alternaria brassicicola. Compared to wild‐type plants, the pmr4‐1 mutant displayed enhanced basal resistance against P. syringae, which correlated with constitutive expression of the PR‐1 gene. Treating the pmr4‐1 mutant with BABA boosted the already elevated levels of PR‐1 gene expression, and further increased the level of resistance. Hence, BABA‐IR against P. syringae does not require PMR4‐derived callose. Conversely, pmr4‐1 plants showed enhanced susceptibility to A. brassicicola, and failed to show BABA‐IR. Wild‐type plants showing BABA‐IR against A. brassicicola produced increased levels of JA. The pmr4‐1 mutant produced less JA upon A. brassicicola infection than the wild‐type. Blocking SA accumulation in pmr4‐1 restored basal resistance, but not BABA‐IR against A. brassicicola. This suggests that the mutant’s enhanced susceptibility to A. brassicicola is caused by SA‐mediated suppression of JA, whereas the lack of BABA‐IR is caused by its inability to produce callose. A. brassicicola infection suppressed ABA accumulation. Pre‐treatment with BABA antagonized this ABA accumulation, and concurrently potentiated expression of the ABA‐responsive ABI1 gene. Hence, BABA prevents pathogen‐induced suppression of ABA accumulation, and sensitizes the tissue to ABA, causing augmented deposition of PMR4‐derived callose

Magnetic tweezers for intracellular applications

doi:10.1063/1.1599066 http://rsi.aip.org/rsinak/v74/i9/p4158_s1We have designed and constructed a versatile magnetic tweezer primarily for intracellular investigations. The micromanipulator uses only two coils to simultaneously magnetize to saturation micron-size superparamagnetic particles and generate high magnitude constant field gradients over cellular dimensions. The apparatus resembles a miniaturized Faraday balance, an industrial device used to measure magnetic susceptibility. The device operates in both continuous and pulse modes. Due to its compact size, the tweezers can conveniently be mounted on the stage of an inverted microscope and used for intracellular manipulations. A built-in temperature control unit maintains the sample at physiological temperatures. The operation of the tweezers was tested by moving 1.28 μm diameter magnetic beads inside macrophages with forces near 500 pN.This work was partially supported by the NSF ~Grant No. DBI-9730999!

β-Aminobutyric Acid-induced Resistance in Plants

Thehe broad sprectrum protective effect of the non-protein amino acid β-aminobutyric acid (BABA) against numerous plant diseases has been well-documented in the literature. Here, we present an overview of BABA-induced protection in various pathosystems. Contriidictory reports concerning the mechanism of action underlying this type of protection incited us to take advantage of Arabidopsis/pathogen interactions as model systems to investigate the action of BABA at the genetic and molecular level. We present evidence that the protective effect of BABA is due to a potentiation of natural defense mechanisms against biotic and abiotic stresses. In order to dissect the pathways involved in potentiation by BABA describe the use of a mutational approach based on BABA-induced female sterility in Arabidopsi

Bioprinting: Development of a novel approach for engineering three-dimensional tissue structures [abstract]

Abstract only availableBioprinting is a tissue engineering technique in which spherical cell aggregates, the "bio-ink", are deposited into biocompatible hydrogels, the "bio-paper", by a 3-axis "bio-printer". The aggregates can be deposited into essentially any 3D configuration, and when comprised of adhesive and motile cells aggregate fusion occurs. This self-organizing, liquid-like nature of these tissues is described on a molecular basis by The Differential Adhesion Hypothesis (DAH). The techniques we have developed are quite unique because of the high degree of automation that has been incorporated into our processes and the variety of engineered tissues that we are capable of creating. Despite automation, the creation of aggregates remains a nontrivial and time intensive process. The entire process of aggregate formation from initial cell culture to mature aggregate ready to be loaded into the printer takes approximately five days. This time is a limiting factor in the potential use of bio-printing as a source of on-demand tissues for clinical applications. A solution to this potential problem lies in the cryopreservation of aggregates. Freezing mediums and freezing protocols were tested and the effect of the freezing process on aggregate fusion was determined. An alternate solution to expedite the bioprinting process could lie in the printing of cell 'sausages', tightly packed cylinders of cells. In this method aggregate preparation is forgone. Elimination of this step could allow for increased time in tissue creation. Cell sausage printing provides another technique that could be incorporated into the fabrication of complex tissues. Our experiments in this novel and developing technology of bioprinting represent steps towards building complex tissues via self-assembly.McNair Scholars Progra

The evolving technology of bio-printing

Abstract only availableBio-printing is a novel method of tissue engineering that uses living cell spheroids as the 'bio-ink' and biocompatible gels as the 'bio-paper' with a three dimensional printer that deposits these aggregates into the gel with great precision. The deposited aggregates fuse into three dimensional tissue structures of the desired conformation due to the liquid like nature of cells and tissues, serving as the driving force of biological self assembly. Successful results from previous experiments and theoretical modeling of the fusion process prompted the development of a standardized and automated method that increases the speed, accuracy and reproducibility of printing. To fulfill these requirements, a cell packer, an aggregate cutter and bio-printer was developed, calibrated and tested. The tools produced more uniform and spherical aggregates as compared to the manual protocols, allowing the standard size and shape necessary for rapid and precise printing. The printed structures (ring and grid-like arrangements of aggregates) fused into toroids and compact sheets, fundamental building blocks of a living organism. The precision of the printing, combined with the cell packer and aggregate cutter makes bio-printing a feasible technology. The automated process using organ specific cells could allow histologically analogous tissues to be produced and used for tissue repair and regeneration.Life Sciences Undergraduate Research Opportunity Progra