Part II: Fibroblasts preferentially migrate in the direction of principal strain

A growing body of evidence suggests that the sensory information from the cytoskeleton and integrins may be responsible for guiding migration during mechano- and haptotaxis. However, the dual function of these subcellular structures as mechano-sensors and -actuators is only partially understood. Using a new cell chamber described in the preceding companion paper (Ref to part I, Raeber etal. 2007a) we investigated the migration response of adhesion-dependent fibroblasts embedded 3-dimensionally within synthetic protease-sensitive poly(ethylene glycol) hydrogels to stepwise and cyclic mechanical loads. To that end, we developed a spatially and temporally resolved migration analysis technique capable of providing estimates of statistical cell migration parameters along and perpendicular to the main strain direction. Fibroblasts reoriented themselves in the direction of principal strain, increased their proteolytic migration activity and moved preferentially parallel to the principal strain axis. These results point to a possible correlation between planes of iso-strain and migration directio

Part II: Fibroblasts preferentially migrate in the direction of principal strain

A growing body of evidence suggests that the sensory information from the cytoskeleton and integrins may be responsible for guiding migration during mechano- and haptotaxis. However, the dual function of these subcellular structures as mechano-sensors and -actuators is only partially understood. Using a new cell chamber described in the preceding companion paper (Ref to part I, Raeber et al. 2007a) we investigated the migration response of adhesion-dependent fibroblasts embedded 3-dimensionally within synthetic protease-sensitive poly(ethylene glycol) hydrogels to stepwise and cyclic mechanical loads. To that end, we developed a spatially and temporally resolved migration analysis technique capable of providing estimates of statistical cell migration parameters along and perpendicular to the main strain direction. Fibroblasts reoriented themselves in the direction of principal strain, increased their proteolytic migration activity and moved preferentially parallel to the principal strain axis. These results point to a possible correlation between planes of iso-strain and migration direction

Sorting live stem cells based on Sox2 mRNA expression.

PMCID: PMC3507951This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner

Living biointerfaces based on non-pathogenic bacteria to direct cell differentiation

Genetically modified Lactococcus lactis, non-pathogenic bacteria expressing the FNIII7-10 fibronectin fragment as a protein membrane have been used to create a living biointerface between synthetic materials and mammalian cells. This FNIII7-10 fragment comprises the RGD and PHSRN sequences of fibronectin to bind α5β1 integrins and triggers signalling for cell adhesion, spreading and differentiation. We used L. lactis strain to colonize material surfaces and produce stable biofilms presenting the FNIII7-10 fragment readily available to cells. Biofilm density is easily tunable and remains stable for several days. Murine C2C12 myoblasts seeded over mature biofilms undergo bipolar alignment and form differentiated myotubes, a process triggered by the FNIII7-10 fragment. This biointerface based on living bacteria can be further modified to express any desired biochemical signal, establishing a new paradigm in biomaterial surface functionalisation for biomedical applications

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications. © 2013 Goh et al

Selective laser melting–enabled electrospinning: Introducing complexity within electrospun membranes

Additive manufacturing technologies enable the creation of very precise and well-defined structures that can mimic hierarchical features of natural tissues. In this article, we describe the development of a manufacturing technology platform to produce innovative biodegradable membranes that are enhanced with controlled microenvironments produced via a combination of selective laser melting techniques and conventional electrospinning. This work underpins the manufacture of a new generation of biomaterial devices that have significant potential for use as both basic research tools and components of therapeutic implants. The membranes were successfully manufactured and a total of three microenvironment designs (niches) were chosen for thorough characterisation. Scanning electron microscopy analysis demonstrated differences in fibre diameters within different areas of the niche structures as well as differences in fibre density. We also showed the potential of using the microfabricated membranes for supporting mesenchymal stromal cell culture and proliferation. We demonstrated that mesenchymal stromal cells grow and populate the membranes penetrating within the niche-like structures. These findings demonstrate the creation of a very versatile tool that can be used in a variety of tissue regeneration applications including bone healing

Hybrid Titanium/Biodegradable Polymer Implants with an Hierarchical Pore Structure as a Means to Control Selective Cell Movement

UNLABELLED: In order to improve implant success rate, it is important to enhance their responsiveness to the prevailing conditions following implantation. Uncontrolled movement of inflammatory cells and fibroblasts is one of these in vivo problems and the porosity properties of the implant have a strong effect on these. Here, we describe a hybrid system composed of a macroporous titanium structure filled with a microporous biodegradable polymer. This polymer matrix has a distinct porosity gradient to accommodate different cell types (fibroblasts and epithelial cells). The main clinical application of this system will be the prevention of restenosis due to excessive fibroblast migration and proliferation in the case of tracheal implants. METHODOLOGY/PRINCIPAL FINDINGS: A microbead-based titanium template was filled with a porous Poly (L-lactic acid) (PLLA) body by freeze-extraction method. A distinct porosity difference was obtained between the inner and outer surfaces of the implant as characterized by image analysis and Mercury porosimetry (9.8±2.2 µm vs. 36.7±11.4 µm, p≤0.05). On top, a thin PLLA film was added to optimize the growth of epithelial cells, which was confirmed by using human respiratory epithelial cells. To check the control of fibroblast movement, PKH26 labeled fibroblasts were seeded onto Titanium and Titanium/PLLA implants. The cell movement was quantified by confocal microscopy: in one week cells moved deeper in Ti samples compared to Ti/PLLA. CONCLUSIONS: In vitro experiments showed that this new implant is effective for guiding different kind of cells it will contact upon implantation. Overall, this system would enable spatial and temporal control over cell migration by a gradient ranging from macroporosity to nanoporosity within a tracheal implant. Moreover, mechanical properties will be dependent mainly on the titanium frame. This will make it possible to create a polymeric environment which is suitable for cells without the need to meet mechanical requirements with the polymeric structure

LEGO® bricks as building blocks for centimeter-scale biological environments

LEGO bricks are commercially available interlocking pieces of plastic that are conventionally used as toys. We describe their use to build engineered environments for cm-scale biological systems, in particular plant roots. Specifically, we take advantage of the unique modularity of these building blocks to create inexpensive, transparent, reconfigurable, and highly scalable environments for plant growth in which structural obstacles and chemical gradients can be precisely engineered to mimic soil

<i>Gastruloids</i> develop the three body axes in the absence of extraembryonic tissues and spatially localised signalling

Establishment of the three body axes is a critical step during animal development. In mammals, genetic studies have shown that a combination of precisely deployed signals from extraembryonic tissues position the anteroposterior axis (AP) within the embryo and lead to the emergence of the dorsoventral (DV) and left-right (LR) axes. We have used Gastruloids , embryonic organoids, as a model system to understand this process and find that they are able to develop AP, DV and LR axes as well as to undergo axial elongation in a manner that mirror embryos. The Gastruloids can be grown for 160 hours and form derivatives from ectoderm, mesoderm and endoderm. We focus on the AP axis and show that in the Gastruloids this axis is registered in the expression of T/Bra at one pole that corresponds to the tip of the elongation. We find that localisation of T/Bra expression depends on the combined activities of Wnt/ β -Catenin and Nodal/Smad2,3 signalling, and that BMP signalling is dispensable for this process. Furthermore, AP axis specification occurs in the absence of both extraembryonic tissues and of localised sources of signalling. Our experiments show that Nodal, together with Wnt/ β -Catenin signalling, is essential for the expression of T/Bra but that Wnt signalling has a separable activity in the elongation of the axis. The results lead us to suggest that, in the embryo, the role of the extraembryonic tissues might not be to induce the axes but to bias an intrinsic ability of the embryo to break its initial symmetry and organise its axes. One sentence summary Culture of aggregates of defined number of Embryonic Stem cells leads to self-organised embryo-like structures which, in the absence of localised signalling from extra embryonic tissues and under the autonomous influence of Wnt and Nodal signalling, develop the three main axes of the body

Anteroposterior polarity and elongation in the absence of extraembryonic tissues and spatially localised signalling in Gastruloids, mammalian embryonic organoids

The establishment of the anteroposterior (AP) axis is a critical step during animal embryo development. In mammals, genetic studies have shown that this process relies on signals spatiotemporally deployed in the extraembryonic tissues that locate the position of the head and the onset of gastrulation, marked by T/Brachyury (T/Bra) at the posterior of the embryo. Here, we use Gastruloids, mESC-based organoids, as a model system to study this process. We find that Gastruloids localise T/Bra expression to one end and undergo elongation similar to the posterior region of the embryo suggesting that they develop an AP axis. This process relies on precisely timed interactions between Wnt/β-Catenin and Nodal signalling, whereas BMP signalling is dispensable. Additionally, polarised T/Bra expression occurs in the absence of extraembryonic tissues or localised sources of signals. We suggest that the role of extraembryonic tissues in the mammalian embryo might not be to induce the axes but to bias an intrinsic ability of the embryo to initially break symmetry. Furthermore, we suggest that Wnt signalling has a separable activity involved in the elongation of the axis.BBSRC (BB/M023370/1), European Commission FP7 ERC Advanced Investigator Grants (AIG) (250316), EPSRC (1359454), NC3Rs (NC/P001467/1