Non-adhesive Regions Research Articles

Effect of Gravity on Bacterial Adhesion to Heterogeneous Surfaces.

Bacterial adhesion is the first step in the formation of surface biofilms. The number of bacteria that bind to a surface from the solution depends on how many bacteria can reach the surface (bacterial transport) and the strength of interactions between bacterial adhesins and surface receptors (adhesivity). By using microfluidic channels and video microscopy as well as computational simulations, we investigated how the interplay between bacterial transport and adhesivity affects the number of the common human pathogen Escherichia coli that bind to heterogeneous surfaces with different receptor densities. We determined that gravitational sedimentation causes bacteria to concentrate at the lower surface over time as fluid moves over a non-adhesive region, so bacteria preferentially adhere to adhesive regions on the lower, inflow-proximal areas that are downstream of non-adhesive regions within the entered compartments. Also, initial bacterial attachment to an adhesive region of a heterogeneous lower surface may be inhibited by shear due to mass transport effects alone rather than shear forces per se, because higher shear washes out the sedimented bacteria. We also provide a conceptual framework and theory that predict the impact of sedimentation on adhesion between and within adhesive regions in flow, where bacteria would likely bind both in vitro and in vivo, and how to normalize the bacterial binding level under experimental set-ups based on the flow compartment configuration.

Mesenchymal Migration on Adhesive-Nonadhesive Alternate Surfaces in Macrophages.

Mesenchymal migration usually happens on adhesive substrates, while cells adopt amoeboid migration on low/nonadhesive surfaces. Protein-repelling reagents, e.g., poly(ethylene) glycol (PEG), are routinely employed to resist cell adhering and migrating. Contrary to these perceptions, this work discovers a unique locomotion of macrophages on adhesive-nonadhesive alternate substrates in vitro that they can overcome nonadhesive PEG gaps to reach adhesive regions in the mesenchymal mode. Adhering to extracellular matrix regions is a prerequisite for macrophages to perform further locomotion on the PEG regions. Podosomes are found highly enriched on the PEG region in macrophages and support their migration across the nonadhesive regions. Increasing podosome density through myosin IIA inhibition facilitates cell motility on adhesive-nonadhesive alternate substrates. Moreover, a developed cellular Potts model reproduces this mesenchymal migration. These findings together uncover a new migratory behavior on adhesive-nonadhesive alternate substrates in macrophages.

Active chemo-mechanical feedbacks dictate the collective migration of cells on patterned surfaces

Recent evidence has demonstrated that, when cultured on micro-patterned surfaces, living cells can move in a coordinated manner and form distinct migration patterns, including flowing chain, suspended propagating bridge, rotating vortex, etc. However, the fundamental question of exactly how and why cells migrate in these fashions remains elusive. Here, we present a theoretical investigation to show that the tight interplay between internal cellular activities, such as chemo-mechanical feedbacks and polarization, and external geometrical constraints are behind these intriguing experimental observations. In particular, on narrow strip patterns, strongly force-dependent cellular contractility and intercellular adhesion were found to be critical for reinforcing the leading edge of the migrating cell monolayer and eventually result in the formation of suspended cell bridges flying over nonadhesive regions. On the other hand, a weak force-contractility feedback led to the movement of cells like a flowing chain along the adhesive strip. Finally, we also showed that the random polarity forces generated in migrating cells are responsible for driving them into rotating vortices on strips with width above a threshold value (~10 times the size of the cell).

Dual Action Antimicrobial Surfaces: Alternating Photopatterns Maintain Contact‐Killing Properties with Reduced Biofilm Formation

AbstractContact‐killing antimicrobial coatings are important for reducing medical device related nosocomial bacterial infections, yet they inadvertently suffer from rapid bacterial colonization. To lessen the extent of biofilm formation on such surfaces, it is hypothesized that coatings containing alternating regions of a low‐fouling polymer incorporated into a contact‐killing surface would reduce bacterial colonization, while still allowing for the contact‐killing properties to be retained. To this end, photopatterned surfaces are developed with alternating regions comprised of a crosslinked low‐fouling zwitterionic copolymer and regions containing the antimicrobial peptide nisin for contact‐killing. The surfaces are characterized by X‐ray photoelectron spectroscopy and water contact angle measurements and assessed for their efficacy against Staphylococcus epidermidis colonization. The dual antimicrobial action surfaces present the synergistic advantages of both classes of coatings against the prolific biofilm‐forming bacterium, reducing the biofilm surface coverage by 70% relative to the nonpatterned control, while still retaining their contact‐killing activity. The results suggest that patterned surfaces, which combine nonadhesive regions with contact killing regions, have the potential to provide improved control over bacterial colonization, biofilm formation, and medical device‐associated infections.

Distance-dependent adhesion of vascular cells on biofunctionalized nanostructures

Abstract Cell-cell and cell-extracellular matrix (ECM) adhesion regulates fundamental cellular functions and is crucial for cell-material contact. Adhesion is influenced by many factors like affinity and specificity of the receptor-ligand interaction or overall ligand concentration and density. To investigate molecular details of cell-ECM and cadherins (cell-cell) interaction in vascular cells functional nanostructured surfaces were used Ligand-functionalized gold nanoparticles (AuNPs) with 6-8 nm diameter, are precisely immobilized on a surface and separated by non-adhesive regions so that individual integrins or cadherins can specifically interact with the ligands on the AuNPs. Using 40 nm and 90 nm distances between the AuNPs and functionalized either with peptide motifs of the extracellular matrix (RGD or REDV) or vascular endothelial-cadherins (VEC), the influence of distance and ligand specificity on spreading and adhesion of endothelial cells (ECs) and smooth muscle cells (SMCs) was investigated. We demonstrate that RGD-dependent adhesion of vascular cells is similar to other cell types and that the distance dependence for integrin binding to ECM-peptides is also valid for the REDV motif. VEC-ligands decrease adhesion significantly on the tested ligand distances. These results may be helpful for future improvements in vascular tissue engineering and for development of implant surfaces.

Dynamics of Cell Ensembles on Adhesive Micropatterns: Bridging the Gap between Single Cell Spreading and Collective Cell Migration.

The collective dynamics of multicellular systems arise from the interplay of a few fundamental elements: growth, division and apoptosis of single cells; their mechanical and adhesive interactions with neighboring cells and the extracellular matrix; and the tendency of polarized cells to move. Micropatterned substrates are increasingly used to dissect the relative roles of these fundamental processes and to control the resulting dynamics. Here we show that a unifying computational framework based on the cellular Potts model can describe the experimentally observed cell dynamics over all relevant length scales. For single cells, the model correctly predicts the statistical distribution of the orientation of the cell division axis as well as the final organisation of the two daughters on a large range of micropatterns, including those situations in which a stable configuration is not achieved and rotation ensues. Large ensembles migrating in heterogeneous environments form non-adhesive regions of inward-curved arcs like in epithelial bridge formation. Collective migration leads to swirl formation with variations in cell area as observed experimentally. In each case, we also use our model to predict cell dynamics on patterns that have not been studied before.

NMR spectroscopy reveals the presence and association of lipids and keratin in adhesive gecko setae.

Lipid and protein aggregates are one of the fundamental materials of biological systems. Examples include cell membranes, insect cuticle, vertebrate epidermis, feathers, hair and adhesive structures known as ‘setae’ on gecko toes. Until recently gecko setae were assumed to be composed entirely of keratin, but analysis of footprints left behind by geckos walking on surfaces revealed that setae include various kinds of lipids. However, the arrangement and molecular-level behavior of lipids and keratin in the setae is still not known. In the present study we demonstrate, for the first time, the use of Nuclear Magnetic Resonance (NMR) spectroscopy techniques to confirm the presence of lipids and investigate their association with keratin in ‘pristine' sheds, or natural molts of the adhesive toe pad and non-adhesive regions of the skin. Analysis was also carried on the sheds after they were ‘delipidized’ to remove surface lipids. Our results show a distribution of similar lipids in both the skin and toe shed but with different dynamics at a molecular level. The present study can help us understand the gecko system both biologically and for design of synthetic adhesives, but the findings may be relevant to the characteristics of lipid-protein interactions in other biological systems.

The Role of Ligand Density in the Binding of Von Willebrand Factor by the Glycoprotein Ib-IX-V Complex in Platelets

The initial arrest of platelets at a wound site requires the binding of glycoprotein Ib-IX-V complex (GPIb-IX-V) to the extracellular matrix protein von Willebrand factor (VWF). Increasing forces on these bonds increases the bond lifetime, known as a catch bond. Recently, we have shown that platelets are able to transmit cytoskeletal forces through the GPIbα subunit of GPIb-IX-V, which binds to the A1 domain of VWF. This provides an internal force on the bond to maintain adhesion in the absence of external forces. Integrin engagement and force transmission are known to require receptor clustering. We therefore investigate whether the clustering of GPIb-IX-V receptor is needed for the transmission of cytoskeletal forces to VWF. We first examined whether GPIb-IX-V clusters formed in spreading platelets. GPIbα-positive punctate structures were visualized by confocal microscopy and correlated with dark regions imaged using interference reflection microscopy (IRM), indicating that GPIb-IX-V forms adhesive contacts that are similar in size to integrin-related focal adhesions in platelets. To investigate the effect of ligand density on platelet spreading, we prepared molecularly-defined adhesive ligand spots, which were separated 28 or 108 nm apart by nonadhesive regions using self-assembling gold nanoparticles. These nanoparticles were coated with A1 domain of VWF. When seeded on nanoparticles with 28 nm spacing, platelets appeared well-spread and formed punctate adhesions. However, when seeded on 108 nm spacing, platelet adhesion was significantly reduced and those platelets that did adhere spread poorly. These findings demonstrate that an upper limit for ligand density exists where platelet adhesion and spreading are impeded. Investigating the effects of ligand spacing will facilitate an understanding of GPIbα clustering in platelet adhesion and spreading, thus providing insight into thrombotic diseases and congenital bleeding disorders.

Tracing Lipids and their Association with Keratin in the Adhesive Gecko Setae by NMR Spectroscopy

The gecko toe is one of the intriguing examples of adhesion in nature. Based on a complex structure of hairy features known as ‘setae’, the toe is composed primarily of keratinous material. Recently however, lipids have been found to be associated with the structure of the adhesive setae. However, the organization and the molecular-level behavior of lipids and keratin in the setae is still not known. Here, we demonstrate the use of Solid and Solution State NMR spectroscopy to detect lipids and understand their association with keratin in the molts termed as ‘sheds’ from the toe pad and the non-adhesive regions of the epidermal skin. Our results show a distribution of similar lipids in both the skin and toe shed but with different dynamics at a molecular level. The study has implications in understanding the organization of materials in nature and provides insight to improve fabrication for synthetic adhesives.

Micropatterned multicolor dynamically adhesive substrates to control cell adhesion and multicellular organization.

We present a novel technique to examine cell–cell interactions and directed cell migration using micropatterned substrates of three distinct regions: an adhesive region, a nonadhesive region, and a dynamically adhesive region switched by addition of a soluble factor to the medium. Combining microcontact printing with avidin–biotin capture chemistry, we pattern nonadhesive regions of avidin that become adhesive through the capture of biotinylated fibronectin. Our strategy overcomes several limitations of current two-color dynamically adhesive substrates by incorporating a third, permanently nonadhesive region. Having three spatially and functionally distinct regions allows for the realization of more complex configurations of cellular cocultures as well as intricate interface geometries between two cell populations for diverse heterotypic cell–cell interaction studies. We can now achieve spatial control over the path and direction of migration in addition to temporal control of the onset of migration, enabling studies that better recapitulate coordinated multicellular migration and organization in vitro. We confirm that cellular behavior is unaltered on captured biotinylated fibronectin as compared to printed fibronectin by examining the cells’ ability to spread, form adhesions, and migrate. We demonstrate the versatility of this approach in studies of migration and cellular cocultures, and further highlight its utility by probing Notch–Delta juxtacrine signaling at a patterned interface.

A Slipping Clutch in Neuronal Growth Cones Revealed by Transient Single Molecule Interactions between Flowing Actin and N-Cadherin Adhesions

The molecular clutch model proposes that the actin motile machinery generated inside cells is coupled to adhesion at the cell membrane, thus generating forces on the substrate and allowing cells to move forward. Many reports have provided evidence for clutch-like behaviors in different cell types and adhesive systems, including integrins and cadherins (Giannone et al., Trends Cell Biol 2009). However, the exact mechanisms underlying the dynamic molecular coupling between the actin retrograde flow and adhesion proteins remain elusive. We previously inferred using optical tweezers that a molecular clutch between the actin flow and N-cadherin adhesions could drive growth cone migration (Bard et al., J Neurosci 2008), but did not achieve a direct visualization of the engagement process. Here, to trigger N-cadherin homophilic adhesions at specific locations, we cultured primary neurons on micropatterned substrates comprising arrays of dots coated with purified N-cadherin, surrounded by a cytophobic background. We then tracked the trajectories of single adhesion and cytoskeletal molecules fused with photo-convertible fluorescent proteins (mEOS2), at the ventral surface of growth cones. N-cadherin and α-catenin were immobilized, while the actin retrograde flow was significantly reduced at N-cadherin coated micro-patterns, compared to non-adhesive regions. Normal actin flow on micro-patterns was restored by expression of a dominant negative N-cadherin construct inhibiting the coupling between endogenous N-cadherin and actin, demonstrating specificity of the process. Strikingly, individual actin trajectories exhibited pauses of the order of seconds selectively on N-cadherin-coated micro-patterns. Thus, at the individual molecular level, clutch engagement is characterized by transient interactions between flowing actin filaments and the immobilized N-cadherin/catenin complex. To our knowledge, this study represents the first direct demonstration of the intrinsic molecular coupling underlying the clutch process in growth cone locomotion.

Analysis of the local organization and dynamics of cellular actin networks

Actin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm.

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.(1) While microcontact printing can be employed to directly print a large number of molecules, including proteins,(2) DNA,(3) and silanes,(4) the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.(5) This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern. The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.(6) These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior(7) to the creation of microelectronics.(8) While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,(9) patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.(10).

Mitochondria and membrane cryoinjury in micropatterned cells: Effects of cell–cell interactions

The maintenance of cell membrane integrity is an absolute minimum criterion for the selection of a successful cryopreservation process; however, it is often used as the sole determinant of cell “viability”. Membrane stresses and strains that develop with cell volume fluctuations are only one component of the overall cellular response to freezing. Damage to organelles resulting from excessive concentration of intracellular solutes and/or the alternation of molecular signalling events may affect post-thaw outcomes. As the low temperature response of cells is affected by the presence of cell–cell interactions, the cryopreservation of tissues and tissue model systems would benefit from a more detailed understanding of the sites and mechanisms of cryoinjury. The purpose of this study was to examine the relationship between mitochondria and plasma membrane damage in frozen micropatterned cells and to identify the role of cell–cell interactions. Madin Darby Canine Kidney cells (MDCK) were micropatterned using a polydimethylsiloxane (PDMS) elastomeric stamp to create non-adhesive regions of agarose on untreated glass substrates. Five different cell arrangements were used to examine the effect of cell–cell contact: single cells, cell doublets, linear arrangement of cells, randomly arranged cells and confluent monolayers. Cells were cooled in a programmable alcohol bath at 1 °C/min to −40 °C after extracellular ice nucleation at −5 °C. Post-thaw plasma membrane integrity and mitochondria depolarization were determined using trypan blue and the lipophilic, cyanine derivative JC-1, respectively. alamarBlue was used to assess the post-thaw metabolic activity of the cell arrangements. We found that the incidence of plasma membrane damage and mitochondria integrity increased with decreasing temperature and was dependent on the degree of cell–cell interaction. Mitochondria damage was evident in cells that displayed intact plasma membranes, however this injury could be reversed in the micropatterned cells that are exposed to suprazero temperatures. The results from this study suggest that the exclusive use of membrane integrity as a measure of cell “viability” does not consider subcellular injury that may contribute to delayed recovery and/or cell death following low temperature exposures.

Force generated by actomyosin contraction builds bridges between adhesive contacts

Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)-coated stripes separated by non-adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non-adhesive regions in a treadmilling network. Inhibition of myosin-II (MII) or Rho-kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin-A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross-links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another.

Integrating Sensing Hydrogel Microstructures into Micropatterned Hepatocellular Cocultures

In this paper we describe a microfabrication-derived approach for defining interactions between distinct groups of cells and integrating biosensors with cellular micropatterns. In this approach, photoresist lithography was employed to micropattern cell-adhesive ligand (collagen I) on silane-modified glass substrates. Poly(ethylene glycol) (PEG) photolithography was then used to fabricate hydrogel microstructures in registration with existing collagen I domains. A glass substrate modified in this manner had three types of micropatterned regions: cell-adhesive collagen I domains, moderately adhesive silanized glass regions, and nonadhesive PEG hydrogel regions. Incubation of this substrate with primary rat hepatocytes or HepG2 cells resulted in attachment of hepatic cells on collagen I domains with no adhesion observed on silane-modified glass regions or hydrogel domains. 3T3 fibroblasts added onto the same surface attached on the glass regions around the hepatocytes, completing the coculture. Significantly, PEG hydrogel microstructures remained free of cells and were used to "fence" hepatocytes from fibroblasts, thus limiting communication between the cell types. We also demonstrated that entrapment of enzyme molecules inside hydrogel microstructures did not compromise nonfouling properties of PEG. Building on this result, horse radish peroxidase-containing hydrogel microstructures were integrated into micropatterned cocultures and were used to detect hydrogen peroxide in the culture medium. The surface micropatterning approach described here may be used in the future to simultaneously define and detect endocrine signaling between two distinct cell types.

Cortical Tension Affects the Spatial Heterogeneity of Clathrin-Coated Pit Dynamics

Clathrin-mediated endocytosis (CME) in mammalian cells is critical for many cellular processes including cell surface receptor down-regulation and nutrient uptake. From analyses of protein interaction networks, the actin polymerization machinery is a modular component within the endocytic interactome. However, the precise role of actin in CME is still under debate. Live cell microscopy has revealed a wide variation in the dynamics of clathrin-coated pits (CCPs). To gain insight of the heterogeneity of CCP dynamics and how cortical actin might influence this heterogeneity, we applied total internal reflection fluorescence microscopy to live cells grown on micro-fabricated substrates patterned with adhesive and non-adhesive regions. Cells on patterns showed overall longer CCP lifetimes compared to cells on chemically uniform surfaces, possibly the result of increased cortical tension. CCP lifetime distributions were also significantly different between adhesive and non-adhesive regions. When the structure of cortical actin is weakened by application of an actin monomer sequestering drug latrunculin A (latA), we found that the CCP lifetimes were homogenized to the level of the non-adherent regions. The decrease in CCP lifetime on adherent regions suggests that cortical actin filaments act as barriers at the adherent surface in CME.

Directional control of cell motility through focal adhesion positioning and spatial control of Rac activation

Local physical interactions between cells and extracellular matrix (ECM) influence directional cell motility that is critical for tissue development, wound repair, and cancer metastasis. Here we test the possibility that the precise spatial positioning of focal adhesions governs the direction in which cells spread and move. NIH 3T3 cells were cultured on circular or linear ECM islands, which were created using a microcontact printing technique and were 1 microm wide and of various lengths (1 to 8 microm) and separated by 1 to 4.5 microm wide nonadhesive barrier regions. Cells could be driven proactively to spread and move in particular directions by altering either the interisland spacing or the shape of similar-sized ECM islands. Immunofluorescence microscopy confirmed that focal adhesions assembled preferentially above the ECM islands, with the greatest staining intensity being observed at adhesion sites along the cell periphery. Rac-FRET analysis of living cells revealed that Rac became activated within 2 min after peripheral membrane extensions adhered to new ECM islands, and this activation wave propagated outward in an oriented manner as the cells spread from island to island. A computational model, which incorporates that cells preferentially protrude membrane processes from regions near newly formed focal adhesion contacts, could predict with high accuracy the effects of six different arrangements of micropatterned ECM islands on directional cell spreading. Taken together, these results suggest that physical properties of the ECM may influence directional cell movement by dictating where cells will form new focal adhesions and activate Rac and, hence, govern where new membrane protrusions will form.

Two‐step cell patterning on planar and complex curved surfaces by precision spraying of polymers

Controlling adhesion of living animal cells plays a key role in biosensor fabrication, drug-testing technologies, basic biological research, and tissue engineering applications. Current techniques for cell patterning have two primary limitations: (1) they require photolithography, and (2) they are limited to patterning of planar surfaces. Here we demonstrate a simple, precision spraying method for both positive and negative patterning of planar and curved surfaces to achieve cell patterns rapidly and reproducibly. In this method, which we call precision spraying (PS), a polymer solution is aerosolized, focused with sheath airflow through an orifice, and deposited on the substrate using a deposition head to create approximately 25 microm sized features. In positive patterning, adhesive molecules, such as laminin or polyethylenimine (PEI) were patterned on polydimethylsiloxane (PDMS) substrates in a single spraying operation. A variety of animal cell types were found to adhere to the adhesive regions, and avoid the non-adhesive (bare PDMS) regions. In negative patterning, hydrophobic materials, such as polytetrafluoroethylene (PTFE) and PDMS, were patterned on glass substrates. Cells then formed patterns on the exposed glass regions and avoided the hydrophobic regions. Cellular patterns were maintained for up to 2 weeks in the presence of serum, which normally fouls non-adhesive regions. Additionally, we found that precision spraying enabled micropatterning of complex-curved surfaces. Our results show that precision spraying followed by cell plating enables rapid and flexible cellular micropatterning in two simple steps.

Geometry of Cell Proliferation

Localized differences in cell proliferation can help sculpt tissues during morphogenesis, leading to the complex structures found in mature tissues. In some cases, however, changes in tissue structure occur before changes in cell proliferation (see Ingber). To investigate the possibility that spatial organization could itself provide a feedback mechanism that helps regulate cell proliferation, Nelson et al. cultured bovine pulmonary artery endothelial cells on a substrate consisting of small fibronectin-coated islands surrounded by nonadhesive regions. Examination of proliferation patterns on islands of different sizes and shapes--or of cells grown on undulating surfaces--revealed characteristic patterns of cell proliferation that depended on the overall geometry. Computational modeling indicated that cell proliferation was greatest in predicted regions of high mechanical stress, a correlation that was confirmed by culturing cells over a micromechanical force sensor array that allowed traction forces to be measured directly. Pharmacological inhibition of Rho kinase, myosin light chain kinase, or nonmuscle myosin II ATPase (to decrease tension generated through the cytoskeleton), or disruption of cadherin-mediated intercellular adhesions with a dominant-negative mutant, attenuated gradients of cell proliferation, whereas expression of a constitutively active RhoA mutant enhanced them. Thus, the authors conclude that tissue geometry, through effects on mechanical stress, plays a role in regulating cell proliferation and tissue growth. C. M. Nelson, R. P. Jean, J. L. Tan, W. F. Liu, N. J. Sniadecki, A. A. Spector, C. S. Chen, Emergent patterns of growth controlled by multicellular form and mechanics. Proc. Natl. Acad. Sci. U.S.A. 102 , 11594-11599 (2005). [Abstract] [Full Text] D. E. Ingber, Mechanical control of tissue growth: Function follows form. Proc. Natl. Acad. Sci. U.S.A. 102 , 11571-11572 (2005). [Full Text]