Investigating the limits of filopodial sensing: a brief report using SEM to image the interaction between 10 nm high nano-topography and fibroblast filopodia

Having the ability to control cell behaviour would be of great advantage in tissue engineering. One method of gaining control over cell adhesion, proliferation, guidance and differentiation is use of topography. Whilst it has be known for some time that cells can be guided by micro‐topography, it is only recently becoming clear that cells will respond strongly to nano‐scale topography. The fact that cells will take cues from their micro‐ and nano‐environment suggests that the cells are in some way ‘spatially aware’. It is likely that cells probe the shape of their surroundings using filopodia, and that this initial filopodia/topography interaction may be critical to down‐stream cell reactions to biomaterials, or indeed, the extracellular matrix. One intriguing question is how small a feature can cells sense? In order to investigate the limits of cell sensing, high‐resolution scanning electron microscopy has been used to simultaneously view cell filopodia and 10 nm high nano‐islands. Fluorescence microscopy has also been used to look at adhesion formation. The results showed distinct filopodial/nano‐island interaction and changes in adhesion morphology.     

Disorganized collagen scaffold interferes with fibroblast mediated deposition of organized extracellular matrix in vitro

Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold‐based and scaffold‐free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS—five donors: average age 34.4) or on a bare polycarbonate membrane (five donors: average age 32.4 controls). The organization and morphology of the two culture systems were compared over the long‐term at 4, 8, and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick‐freeze/deep‐etch electron microscopy. Both the scaffold‐free and the collagen‐based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell‐derived matrix in the scaffold‐based constructs was significantly lower than that produced by scaffold‐free constructs (controls). We also observed significant variability in the remodeling of RCS scaffold by PHCFCs. PHCFCs which penetrated the RCS scaffold did exert robust local control over secreted collagen but did not appear to globally reorganize the scaffold effectively in the time period of the study. Consistent with our hypothesis, the results demonstrate that the presence of the scaffold appears to interfere with the global organization of the cell‐derived matrix. The production of highly organized local matrix by fibroblasts which penetrated the scaffold suggests that there is a mechanism which operates close to the cell membrane capable of controlling fibril organization. Nonetheless, the local control of the collagen alignment produced by cells within the scaffold was not continuous and did not result in overall global organization of the construct. Using a disorganized scaffold as a guide to produce highly organized tissue has the potential to delay the production of useful matrix or prevent uniform remodeling. The results of this study may shed light on the recent attempts to use disorganized collagenous matrix as a temporary corneal replacement in vivo which led to a variable remodeling response. Biotechnol. Bioeng. 2012; 109: 2683–2698. © 2012 Wiley Periodicals, Inc.     

Electrospun triple-layered PLLA/gelatin. PRGF/PLLA scaffold induces fibroblast migration

The function of fibroblast cells in wounded areas results in reconstruction of the extra cellular matrix and consequently resolution of granulation tissue. It is suggested that the use of platelet-rich plasma can accelerate the healing process in nonhealing or slow-healing wounds. In this study, a simple and novel method has been used to fabricate an electrospun three-layered scaffold containing plasma rich in growth factor with the aim of increasing the proliferation and migration of fibroblast cells in vitro. First, plasma rich in growth factor was derived from platelet rich plasma, and then a three-layered scaffold was fabricated using PLLA nanofibers as the outer layers and plasma rich in growth factor-containing gelatin fibers as the internal layer. The growth morphology of cells seeded on this scaffold was compared to those seeded on one layered PLLA scaffold. The study of the cell growth rate on different substrates and the migration of cells in response to the drug release of multilayered scaffold was investigated by the cell quantification assay and a modified under agarose assay. Scanning electron microscopy and fluorescence images showed that cells seeded on multilayered scaffold were completely oriented 72 hours after seeding compared to those seeded on PLLA scaffold. The cell quantification assay also indicated significant increase in proliferation rate of cells seeded on three-layered scaffold compared to those seeded on PLLA scaffold and finally, monitoring cell migration proved that cells migrate significantly toward the three-layered scaffold up to 48 to 72 hours and afterwards start to show a diminished migration rate toward this scaffold.     

Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices

The cell seeding density and spatial distribution in a 3-D scaffold are critical to the morphogenetic development of an engineered tissue. A dynamic depth-filtration seeding method was developed to improve the initial cell seeding density and spatial distribution in 3-D nonwoven fibrous matrices commonly used as tissue scaffolds. In this work, trophoblast-like ED27 cells were seeded in poly(ethylene terephthalate) (PET) matrices with various porosities (0.85-0.93). The effects of the initial concentration of cells in the suspension used to seed the PET matrix and the pore size of the matrix on the resulting seeding density and subsequent cell proliferation and tissue development were studied. Compared to the conventional static seeding method, the dynamic depth-filtration seeding method gave a significantly higher initial seeding density (2-4 x 10(7) vs 4 x 10(6) cells/cm3), more uniform cell distribution, and a higher final cell density in the tissue scaffold. The more uniform initial cell spatial distribution from the filtration seeding method also led to more cells in S phase and a prolonged proliferation period. However, both uniform spatial cell distribution and the pore size of the matrices are important to cell proliferation and morphological development in the seeded tissue scaffold. Large-pore matrices led to the formation of cell aggregates and thus might reduce cell proliferation. The dynamic depth-filtration seeding method is better in providing a higher initial seeding density and more uniform cell distribution and is easier to apply to large tissue scaffolds. A depth-filtration model was also developed and can be used to simulate the seeding process and to predict the maximum initial seeding densities in matrices with different porosities.     

Evaluating the biodegradability of Gelatin/Siloxane/Hydroxyapatite (GS-Hyd) complex in vivo and its ability for adhesion and proliferation of rat bone marrow mesenchymal stem cells

Recent studies have shown that the use of biomaterials and new biodegradable scaffolds for repair or regeneration of damaged tissues is of vital importance. Scaffolds used in tissue engineering should be biodegradable materials with three-dimensional structures which guide the growth and differentiation of the cells. They also tune physical, chemical and biological properties for efficient supplying of the cells to the selected tissues and have proper porosity along with minimal toxic effects. In this manner, the study of these characteristics is a giant stride towards scaffold design. In this study, Gelatin/Siloxane/Hydroxyapatite (GS-Hyd) scaffold was synthesized and its morphology, in vivo biodegradability, cytotoxic effects and ability for cell adhesion were investigated using mesenchymal stem cells (MSCs). The cells were treated with different volumes of the scaffold suspension for evaluation of its cytotoxic effects. The MSCs were also seeded on scaffolds and cultured for 2 weeks to evaluate the ability of the scaffold in promoting of cell adhesion and growth. To check the biodegradability of the scaffold in vivo, scaffolds were placed in the rat body for 21 days in three different positions of thigh muscle, testicle, and liver and they were analyzed by scanning electron microscopy (SEM) and weight changes. According to the results of the viability of this study, no cytotoxic effects of GS-Hyd scaffold was found on the cells and MSCs could adhere on the scaffold with expanding their elongations and forming colonies. The rate of degradation as assessed by weight loss was significant within each group along with significant differences between different tissues at the same time point. SEM micrographs also indicated the obvious morphological changes on the surface of the particles and diameter of the pores through different stages of implantation. The greatest amount of degradation happened to the scaffold particles implanted into the muscle, followed by testicle and liver, respectively.     

Regulation of adipose‐tissue‐derived stromal cell orientation and motility in 2D‐ and 3D‐cultures by direct‐current electrical field

Cell alignment and motility play a critical role in a variety of cell behaviors, including cytoskeleton reorganization, membrane‐protein relocation, nuclear gene expression, and extracellular matrix remodeling. Direct current electric field (EF) in vitro can direct many types of cells to align vertically to EF vector. In this work, we investigated the effects of EF stimulation on rat adipose‐tissue‐derived stromal cells (ADSCs) in 2D‐culture on plastic culture dishes and in 3D‐culture on various scaffold materials, including collagen hydrogels, chitosan hydrogels and poly(L‐lactic acid)/gelatin electrospinning fibers. Rat ADSCs were exposed to various physiological‐strength EFs in a homemade EF‐bioreactor. Changes of morphology and movements of cells affected by applied EFs were evaluated by time‐lapse microphotography, and cell survival rates and intracellular calcium oscillations were also detected. Results showed that EF facilitated ADSC morphological changes, under 6 V/cm EF strength, and that ADSCs in 2D‐culture aligned vertically to EF vector and kept a good cell survival rate. In 3D‐culture, cell galvanotaxis responses were subject to the synergistic effect of applied EF and scaffold materials. Fast cell movement and intracellular calcium activities were observed in the cells of 3D‐culture. We believe our research will provide some experimental references for the future study in cell galvanotaxis behaviors.     

Cell colonization in degradable 3D porous matrices

Cell colonization is an important in a wide variety of biological processes and applications including vascularization, wound healing, tissue engineering, stem cell differentiation and biosensors. During colonization porous 3D structures are used to support and guide the ingrowth of cells into the matrix. In this review, we summarize our understanding of various factors affecting cell colonization in three-dimensional environment. The structural, biological and degradation properties of the matrix all play key roles during colonization. Further, specific scaffold properties such as porosity, pore size, fiber thickness, topography and scaffold stiffness as well as important cell material interactions such as cell adhesion and mechanotransduction also influence colonization.Key words: colonization, pore size, porosity, topography, mechanotransduction, degradation, matrix turnover     

The extracellular topographical environment influences ovarian cancer cell behaviour

The importance of the biophysical cellular environment in cancer development has been increasingly recognised but so far has been only superficially studied. Here we investigated the effect of cell-like substrate topography on ovarian cancer cell behaviour and potential underlying signalling pathways. We observed changes in cell morphology in response to substrate topography, which implies modification of structure-function associations. Differences in focal adhesion signalling and Rho/ROCK activity suggested their involvement in the biomechanically-driven cellular responses. Cell-like topography was also shown to modulate the MAPK pathway and hence potentially regulate cell proliferation. The selective regulation of the cells by the mechanotransduction pathways that we noted has wide ranging implications for understanding cancer development. We established that the physical architecture of cell culture substrate is sufficient to influence cancer cell behaviour, independent of genetic composition or biochemical milieu.     

Improvement of biomaterials used in tissue engineering by an ageing treatment

Biomaterials based on crosslinked sponges of biopolymers have been extensively used as scaffolds to culture mammal cells. It is well known that single biopolymers show significant change over time due to a phenomenon called physical ageing. In this research, it was verified that scaffolds used for skin tissue engineering (based on gelatin, chitosan and hyaluronic acid) express an ageing-like phenomenon. Treatments based on ageing of scaffolds improve the behavior of skin-cells for tissue engineering purposes. Physical ageing of dry scaffolds was studied by differential scanning calorimetry and was modeled with ageing kinetic equations. In addition, the physical properties of wet scaffolds also changed with the ageing treatments. Scaffolds were aged up to 3 weeks, and then skin-cells (fibroblasts) were seeded on them. Results indicated that adhesion, migration, viability, proliferation and spreading of the skin-cells were affected by the scaffold ageing. The best performance was obtained with a 2-week aged scaffold (under cell culture conditions). The cell viability inside the scaffold was increased from 60 % (scaffold without ageing treatment) to 80 %. It is concluded that biopolymeric scaffolds can be modified by means of an ageing treatment, which changes the behavior of the cells seeded on them. The ageing treatment under cell culture conditions might become a bioprocess to improve the scaffolds used for tissue engineering and regenerative medicine.     

Nature of thrombin-induced sustained increase in cytosolic calcium concentration in cultured endothelial cells

It has recently been appreciated that thrombin induces the retraction of endothelial cells resulting in an alteration of the integrity of the monolayers. We studied thrombin-induced changes in cytosolic calcium concentration (Ca2+i) using microfluorometry of fura-2-loaded single cells, cell topography (scanning electron microscopy), and cytoskeleton (rhodamine phalloidin) in endothelial cells. Thrombin caused an initial and sustained phase of an increase in Ca2+i. Pretreatment with pertussis toxin abolished both phases of Ca2+i response. Sustained phase of thrombin effect required extracellular calcium. Pretreatment of endothelial cells with indomethacin protracted the sustained phase, whereas a lipoxygenase inhibitor, nordihydroguaiaretic acid curtailed it. Thrombin caused a marked retraction of confluent endothelial cells coincident with the sustained phase of Ca2+i response. This was paralleled by the formation of gaps in F-actin distribution at the periphery of the cells. Pretreatment of endothelial cells with nordihydroguaiaretic acid blunted the thrombin-induced cell retraction. Microinjection of various putative messengers into the endothelial cells showed that initial Ca2+ mobilization is not sufficient to account for sustained elevation of Ca2+i. The sustained response required microinjection of phospholipase A2 or co-injection of phospholipase A2 with phosphatidylinositol 4,5-bisphosphate-specific phospholipase C, phosphatidylinositol 1,4,5-trisphosphate, or CaCl2, further implying that thrombin receptor(s) can be coupled to both phospholipases C and A2. Sustained elevation of Ca2+i was a necessary prerequisite for the thrombin-induced changes in endothelial cell topography.     

Nanotopographical control of human osteoprogenitor differentiation

Current load-bearing orthopaedic implants are produced in 'bio-inert' materials such as titanium alloys. When inserted into the reamed bone during hip or knee replacement surgery the implants interact with mesenchymal populations including the bone marrow. Bio-inert materials are shielded from the body by differentiation of the cells along the fibroblastic lineage producing scar tissue and inferior healing. This is exacerbated by implant micromotion, which can lead to capsule formation. Thus, next-generation implant materials will have to elicit influence over osteoprogenitor differentiation and mesenchymal populations in order to recruit osteoblastic cells and produce direct bone apposition onto the implant. A powerful method of delivering cues to cells is via topography. Micro-scale topography has been shown to affect cell adhesion, migration, cytoskeleton, proliferation and differentiation of a large range of cell types (thus far all cell types tested have been shown to be responsive to topographical cues). More recent research with nanotopography has also shown a broad range of cell response, with fibroblastic cells sensing down to 10 nm in height. Initial studies with human mesenchymal populations and osteoprogenitor populations have again shown strong cell responses to nanofeatures with increased levels of osteocalcin and osteopontin production from the cells on certain topographies. This is indicative of increased osteoblastic activity on the nanotextured materials. Looking at preliminary data, it is tempting to speculate that progenitor cells are, in fact, more responsive to topography than more mature cell types and that they are actively seeking cues from their environment. This review will investigate the range of nanotopographies available to researchers and our present understanding of mechanisms of progenitor cell response. Finally, it will make some speculations of the future of nanomaterials and progenitor cells in tissue engineering.     

Effects of Different Tissue Microenvironments on Gene Expression in Breast Cancer Cells

In metastasis, circulating tumor cells penetrate the walls of blood vessels and enter the metastatic target tissue, thereby becoming exposed to novel and relatively unsupportive microenvironments. In the new microenvironments, the tumor cells often remain in a dormant state indefinitely and must adapt before they are able to successfully colonize the tissue. Very little is known about this adaptive process. We studied temporal changes in gene expression when breast cancer cells adapt to survive and grow on brain, bone marrow, and lung tissue maintained in an in vivo culture system, as models of the metastatic colonization of these tissues. We observed the transient activation of genes typically associated with homeostasis and stress during the initial stages of adaptation, followed by the activation of genes that mediate more advanced functions, such as elaboration of cell morphology and cell division, as the cells adapted to thrive in the host tissue microenvironment. We also observed the temporary induction of genes characteristic of the host tissue, which was particularly evident when tumor cells were grown on brain tissue. These early transient gene expression events suggest potential points of therapeutic intervention that are not evident in data from well-established tumors.     

Cell colonization in degradable 3D porous matrices

Cell colonization is an important in a wide variety of biological processes and applications including vascularization, wound healing, tissue engineering, stem cell differentiation, and biosensors. During colonization porous 3D structures are used to support and guide the ingrowth of cells into the matrix. In this review, we summarize our understanding of various factors affecting cell colonization in 3-dimensional environment. The structural, biological, and degradation properties of the matrix all play key roles during colonization. Further, specific scaffold properties such as porosity, pore size, fiber thickness, topography, and scaffold stiffness as well as important cell material interactions such as cell adhesion and mechanotransduction also influence colonization.     

Label-free, high-throughput measurements of dynamic changes in cell nuclei using angle-resolved low coherence interferometry

Accurate measurements of nuclear deformation, i.e., structural changes of the nucleus in response to environmental stimuli, are important for signal transduction studies. Traditionally, these measurements require labeling and imaging, and then nuclear measurement using image analysis. This approach is time-consuming, invasive, and unavoidably perturbs cellular systems. Light scattering, an emerging biophotonics technique for probing physical characteristics of living systems, offers a promising alternative. Angle-resolved low-coherence interferometry (a/LCI), a novel light scattering technique, was developed to quantify nuclear morphology for early cancer detection. In this study, a/LCI is used for the first time to noninvasively measure small changes in nuclear morphology in response to environmental stimuli. With this new application, we broaden the potential uses of a/LCI by demonstrating high-throughput measurements and by probing aspherical nuclei. To demonstrate the versatility of this approach, two distinct models relevant to current investigations in cell and tissue engineering research are used. Structural changes in cell nuclei due to subtle environmental stimuli, including substrate topography and osmotic pressure, are profiled rapidly without disrupting the cells or introducing artifacts associated with traditional measurements. Accuracy ≥ 3% is obtained for the range of nuclear geometries examined here, with the greatest deviations occurring for the more complex geometries. Given the high-throughput nature of the measurements, this deviation may be acceptable for many biological applications that seek to establish connections between morphology and function.     

Structural properties of scaffolds: Crucial parameters towards stem cells differentiation

Tissue engineering is a multidisciplinary field that applies the principles of engineering and life-sciences for regeneration of damaged tissues. Stem cells have attracted much interest in tissue engineering as a cell source due to their ability to proliferate in an undifferentiated state for prolonged time and capability of differentiating to different cell types after induction. Scaffolds play an important role in tissue engineering as a substrate that can mimic the native extracellular matrix and the properties of scaffolds have been shown to affect the cell behavior such as the cell attachment, proliferation and differentiation. Here, we focus on the recent reports that investigated the various aspects of scaffolds including the materials used for scaffold fabrication, surface modification of scaffolds, topography and mechanical properties of scaffolds towards stem cells differentiation effect. We will present a more detailed overview on the effect of mechanical properties of scaffolds on stem cells fate.     

聚己内酯静电纺丝纳米纤维支架用于小鼠诱导多能干细胞培养

干细胞联合生物支架材料体外构建功能性组织与器官,成为当前组织再生研究的重要策略,而探求具有良好生物相容性的支架材料是其关键.本研究采用扫描电镜、噻唑蓝（MTT）法、荧光显微染色等方法检测小鼠诱导多能干细胞（murine induced pluripotent stem cells, miPSCs）在聚己内酯（poly ε-caprolactone, PCL）静电纺丝纳米纤维支架上的粘附、增殖等生物学特性,探究聚己内酯纳米纤维支架与miPSCs的生物相容性. 结果显示,miPSC在PCL纳米纤维支架上具有良好粘附性并呈集落样生长,其增殖能力及干性标记物（Oct4-GFP+）的表达均不亚于标准对照组;扫描电镜显示,miPSC在PCL纳米纤维支架材料上呈现出绒毛状突起的表面结构.上述结果表明,PCL纳米纤维支架可促进miPSCs的粘附、自我增殖以及干性维持,两者具有良好的生物相容性,为下一步联合生物支架材料与干细胞构建功能性组织奠定了基础.     

Biological matrices and bionanotechnology

A crucial step towards the goal of tissue engineering a heart valve will be the choice of scaffold onto which an appropriate cell phenotype can be seeded. Successful scaffold materials should be amenable to modification, have a controlled degradation, be compatible with the cells, lack cytotoxicity and not elicit an immune or inflammatory response. In addition, the scaffold should induce appropriate responses from the cells seeded onto it, such as cell attachment, proliferation and remodelling capacity, all of which should promote the formation of a tissue construct that can mimic the structure and function of the native valve. This paper discusses the various biological scaffolds that have been considered and are being studied for use in tissue engineering a heart valve. Also, strategies to enhance the biological communication between the scaffold and the cells seeded onto it as well as the use of bionanotechnology in the manufacture of scaffolds possessing the desired properties will be discussed.     

Influence of flow rate and scaffold pore size on cell behavior during mechanical stimulation in a flow perfusion bioreactor

Mechanically stimulating cell-seeded scaffolds by flow-perfusion is one approach utilized for developing clinically applicable bone graft substitutes. A key challenge is determining the magnitude of stimuli to apply that enhances cell differentiation but minimizes cell detachment from the scaffold. In this study, we employed a combined computational modeling and experimental approach to examine how the scaffold mean pore size influences cell attachment morphology and subsequently impacts upon cell deformation and detachment when subjected to fluid-flow. Cell detachment from osteoblast-seeded collagen-GAG scaffolds was evaluated experimentally across a range of scaffold pore sizes subjected to different flow rates and exposure times in a perfusion bioreactor. Cell detachment was found to be proportional to flow rate and inversely proportional to pore size. Using this data, a theoretical model was derived that accurately predicted cell detachment as a function of mean shear stress, mean pore size, and time. Computational modeling of cell deformation in response to fluid flow showed the percentage of cells exceeding a critical threshold of deformation correlated with cell detachment experimentally and the majority of these cells were of a bridging morphology (cells stretched across pores). These findings will help researchers optimize the mean pore size of scaffolds and perfusion bioreactor operating conditions to manage cell detachment when mechanically simulating cells via flow perfusion.     

Using continuous porous silicon gradients to study the influence of surface topography on the behaviour of neuroblastoma cells

The effects of surface topography on cell behaviour are the subject of intense research in cell biology. These effects have so far only been studied using substrate surfaces of discretely different topography. In this paper, we present a new approach to characterise cell growth on porous silicon gradients displaying pore sizes from several thousands to a few nanometers. This widely applicable format has the potential to significantly reduce sample numbers and hence analysis time and cost. Our gradient format was applied here to the culture of neuroblastoma cells in order to determine the effects of topography on cell growth parameters. Cell viability, morphology, length and area were characterised by fluorescence and scanning electron microscopy. We observed a dramatic influence of changes in surface topography on the density and morphology of adherent neuroblastoma cells. For example, pore size regimes where cell attachment is strongly discouraged were identified providing cues for the design of low-fouling surfaces. On pore size regimes more conducive to cell attachment, lateral cell-cell interactions crosslinked the cell layer to the substratum surface, while direct substrate-cell interactions were scarce. Finally, our study revealed that cells were sensitive to nanoscale surface topography with feature sizes of <20 nm.