Heading in the right direction : guiding cellular alignment by
substrate anisotropy
Citation for published version (APA):
Buskermolen, A. B. C. (2018). Heading in the right direction : guiding cellular alignment by substrate anisotropy. Technische Universiteit Eindhoven.
Document status and date: Published: 03/12/2018 Document Version:
Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication:
• A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website.
• The final author version and the galley proof are versions of the publication after peer review.
• The final published version features the final layout of the paper including the volume, issue and page numbers.
Link to publication
General rights
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain
• You may freely distribute the URL identifying the publication in the public portal.
If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement:
www.tue.nl/taverne
Take down policy
If you believe that this document breaches copyright please contact us at: openaccess@tue.nl
Guiding cellular alignment by substrate anisotropy
ISBN: 978-94-6380-106-5
Copyright© 2018by A.B.C. Buskermolen
All rights reserved. No part of this book may be reproduced, stored in a database or retrieval system, or published, in any form or in any way, elec-tronically, mechanically, by print, by photo print, microfilm or any other means without prior written permission by the author.
Cover design: painting by A.B.C. Buskermolen and Jasmijn Felix
This document was typeset using the typographical look-and-feelclassicthesis developed by André Miede.
Printed by ProefschriftMaken | www.proefschriftmaken.nl
Financial support by the Dutch Heart foundation for the publication of this thesis is gratefully acknowledged. The research described in this thesis was supported by a grant of the Dutch Heart Foundation CVON 2012-01 1Valve.
Heading in the right direction
Guiding cellular alignment by
substrate anisotropy
PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de
Technische Universiteit Eindhoven, op gezag van de
rector magnificus prof.dr.ir. F.P.T. Baaijens, voor een
commissie aangewezen door het College voor
Promoties, in het openbaar te verdedigen op
maandag 3 december 2018 om 11:00 uur
door
Antonetta Brigitte Cornelia Buskermolen
voorzitter: Prof.dr.ir. F.N. van de Vosse 1e promotor: Prof.dr. C.V.C. Bouten co-promotor: Dr. N.A. Kurniawan
leden: Dr. ir. T.F.A. de Greef
Prof.dr.ir. P. Jonkheijm (University of Twente) Prof.dr. G. Koenderink (AMOLF)
Prof.dr. C. Storm
Het onderzoek of ontwerp dat in dit proefschrift wordt beschreven is uitge-voerd in overeenstemming met de TU/e Gedragscode Wetenschapsbeoefe-ning.
Heading in the right direction.
Guiding cellular alignment by substrate anisotropy
Most biological tissues shown an intriguing anisotropic behavior that is driven by the intrinsic structural organization. The orientation of cells plays a central role in the anisotropic micro-architecture of tissues, dictating their biological and mechanical functioning. The cellular orientation response is induced by the specific organization of the structural subcellular components consisting of focal adhesions (FAs) physically linking the extracellular envi-ronment via the actin cytoskeleton to the nucleus. The central aim of this thesis was to get mechanistic insights on how cells sense and respond to substrate anisotropy as a manner to control cellular alignment.
In vivo, the extracellular environment provides anisotropic physical, biochem-ical, and mechanical cues that have an influence on the cellular orientation response. It has been shown that cells prefer to align in the direction of the anisotropy of the environment, a phenomenon referred to as contact guidance. Although intense efforts have been devoted on understanding the mechanisms of contact guidance, surprisingly little is known about the un-derlying role of each individual subcellular component regulating this behav-ior. This knowledge is of fundamental importance for designing strategies for controlling cell behavior towards the restoration of tissue functionality and mechanical integrity.
An unbiased, quantitative evaluation of the intracellular structural compo-nents in conjunction with the morphologoy of cells can aid in the further uncovering of the mechanisms underlying contact guidance. We developed a fast, user-friendly and automated image analysis algorithm capable of cap-turing and characterizing these individual components with a high level of accuracy. The robustness and applicability of the algorithm was demon-strated by the quantification of the morphological changes in response to a variety of environmental changes as well as manipulations of cellular com-ponents of mechanotransductions.
Various micropatterning approaches using two-dimensional (2D) substrates have been developed to study cellular contact guidance to simplify the highly complex environments in vivo. We used microcontact printing to create stripes of fibronectin of varying widths ranging from >1.5 mm to 50 µm. By cultur-ing cells on these patterns, we were able to examine the onset of contact guidance. Surprisingly, we observed that contact guidance emerges at stripe widths much greater than the cell size. To understand this counter-intuitive observation, we combined morphometric analysis of cells and their intra-cellular components with a novel statistical thermodynamics framework for modeling the fluctuating response of cells. This comparison revealed that contact guidance arises from the tendency of cells to maximize their mor-phological entropy through shape fluctuations.
As a next step we mimicked the native-like fibrallar extracellular environ-ment by patterning multiple lines of varying widths and inter-line spacings ranging from 2 µm (FAs) to 200 µm (single cell) to understand the origin of contact guidance. Our data demonstrated two distinct regimes of cellular orientation responses governed by line width. Surprisingly, the cellular con-tact guidance response was lost at smaller line widths, even when individual FAs were spatially constrained. To understand the reason for this unexpected finding, we combined the experimental data with a statistical thermodynam-ics model. This comparison demonstrated that the inter-line spacing, rather than the line width, controlled cell morphology and alignment, suggesting that contact guidance emerges from the energetic tendency of cells to mini-mize the number of non-adhesive gaps to bridge.
To mimic the complexity of the natural micro-environment of cells more closely, a combination of individual controlled cues was applied to study the effects on the cellular orientation response. We provided a detailed de-scription on how to perform protein micropatterning (contact guidance cues) on curved substrates (curvature guidance cues) by using a contactless and maskless UV projection system. We showed that with this approach it was possible to create protein patterns that completely covered curved surfaces of various sizes. This method could then be used to systematically study the cellular responses towards contact guidance and curvature guidance cues.
Taken together, the results of this thesis improve mechanistic understand-ing on the role of substrate anisotropy on the cellular orientation response. More specifically, the obtained fundamental knowledge emphasize that cel-lular alignment is driven by entropy maximization and energy minimization that determines the specific organization of the intracellular components.
i b a c k g r o u n d 1
1 g e n e r a l i n t r o d u c t i o n 3
1.1 Introduction . . . 4 1.2 The structural interaction between the extracellular
environment and the intracellular components . . . 5 1.3 Understanding the influence of anisotropic
environments on the cellular orientation response . . . 8 1.3.1 Manipulating substrate anisotropy . . . 8 1.3.2 Understanding the cellular response to substrate
ani-sotropy . . . 11 1.4 Thesis outline . . . 12 2 u n d e r s ta n d i n g c e l l u l a r o r i e n tat i o n r e s p o n s e s t o c o m
-p l e x b i o -p h y s i c a l e n v i r o n m e n t s 15
2.1 Introduction . . . 16 2.2 The structural mechanotransduction pathway: A
physical connection between the extracellular
environment and the genome . . . 18 2.2.1 Interconnection between the extracellular environment
and the actin
cytoskeleton . . . 19 2.2.2 Interconnection between the actin cytoskeleton and the
nucleus . . . 20 2.3 Cellular orientation response to substrate anisotropy and cyclic
strain . . . 24 2.3.1 Cellular orientation response to substrate anisotropy . . 24 2.3.2 Cellular orientation response to uniaxial cyclic strain . . 32 2.3.3 Cellular orientation response to combined substrate
an-isotropy and uniaxial cyclic strain . . . 39 2.4 Summary and Outlook . . . 43
ii q ua n t i f i c at i o n m e t h o d t o a na ly z e t h e (sub)cellular
o r i e n ta t i o n r e s p o n s e 47
3 a n au t o m at e d q ua n t i tat i v e a na ly s i s o f c e l l, nucleus
a n d f o c a l a d h e s i o n m o r p h o l o g y 49
3.1 Introduction . . . 50
3.2 Materials and methods . . . 52
3.2.1 Experimental design . . . 52
3.2.2 Image analysis algorithm . . . 54
3.3 Results and Discussion . . . 58
3.3.1 Validation of automated detection of cells, nuclei and focal adhesions . . . 58
3.3.2 Robustness and applicability of the algorithm . . . 61
3.3.3 ROCK inhibitor Y-27632 affects FA morphology in a dose-dependent manner . . . 64
3.3.4 Limitations of the algorithm . . . 67
3.4 Conclusion . . . 67
iii t h e i n f l u e n c e o f s u b s t r at e a n i s o t r o p y o n t h e c e l l u -l a r o r i e n tat i o n r e s p o n s e 69 4 e n t r o p i c f o r c e s d r i v e c e l l u l a r c o n ta c t g u i d a n c e 71 4.1 Introduction . . . 72
4.2 Materials and Methods . . . 74
4.2.1 Preparation of micropatterned substrates . . . 74
4.2.2 Cell culture . . . 75
4.2.3 Cell fixation and staining . . . 75
4.2.4 Image analysis . . . 76
4.2.5 Material parameters for myofibroblasts . . . 76
4.3 Results . . . 77
4.3.1 Cell alignment increases with decreasing stripe width . 77 4.3.2 Homeostatic mechanics predictions reproduce morpho-metric observations . . . 82
4.3.3 Emergence of two regimes of contact guidance: wide vs. narrow stripes . . . 83
4.3.4 Regime I: Entropic alignment of cells for stripe widths larger than the cell size . . . 84
4.3.5 Regime II: Biochemical changes accompany alignment at small stripe widths . . . 86
4.3.6 Thermodynamic forces help delineate the two regimes
of alignment . . . 89
4.3.7 Orientational alignment is a consequence of the ten-dency of cells to maximize morphological disorder . . . 92
4.4 Discussion . . . 95
5 c o n ta c t g u i d a n c e r e s u lt s f r o m g a p av o i d a n c e 99 5.1 Introduction . . . .100
5.2 Material and Methods . . . .101
5.2.1 Experiments . . . .101
5.2.2 Modeling . . . .104
5.3 Results . . . .108
5.3.1 Cellular alignment is controlled by substrate anisotropy at submicron to cell size-scales . . . .108
5.3.2 Constrained focal adhesions do not guide cellular align-ment . . . .111
5.3.3 Cells minimize the number of gaps to bridge . . . .115
5.3.4 Increasing the non-adhesive gaps between lines induces cellular alignment . . . .118 5.4 Discussion . . . .123 6 g e n e r at i n g p r o t e i n m i c r o pat t e r n s o n c u r v e d s u b s t r at e s t o s t u d y t h e c o m b i n e d e f f e c t o f c o n ta c t g u i d a n c e a n d c u r vat u r e g u i d a n c e 127 6.1 Introduction . . . .128 6.1.1 Relevance . . . .130
6.1.2 Comparison with other methods, novelty, and advan-tages . . . .131 6.2 Experimental Design . . . .134 6.3 Materials . . . .137 6.4 Procedure . . . .140 6.5 Results . . . .147 6.6 Current limitations . . . .150
6.7 Conclusion and Outlook . . . .156
iv f u t u r e p e r s p e c t i v e s 159 7 d i s c u s s i o n a n d o u t l o o k 161 7.1 Introduction . . . .162
7.2 Main Findings . . . .162
7.3.1 Moving towards in-depth understanding . . . .166 7.3.2 Moving towards complex environments . . . .170 7.4 Conclusion . . . 172 v a p p e n d i x 173 a a p p e n d i x 175 a.1 Appendix Chapter 3 . . . .175 a.2 Appendix Chapter 4 . . . .178 a.3 Appendix Chapter 5 . . . .182 b i b l i o g r a p h y 184 s a m e n vat t i n g 219 a c k n o w l e d g m e n t s 221 c u r r i c u l u m v i ta e 225 p u b l i c at i o n s 227
1D One-dimensional 2D Two-dimensional
2.5D Two-and-a-half-dimensional 3D Three-dimensional
4D Four-dimensional
APTES 3’aminopropyltriethoxy silane
DAPI 4’,6-diamidino-2-phenylindole
DMEM Dulbecco’s Modified Eagles Medium
DMD Digital micro-mirror device
DMSO Dimethyl sulfoxide
ECM Extracellular matrix
FBS Fetal bovine serum
FN Fibronectin
FA Focal adhesion
FAK Focal adhesion kinase
FITC Fluorescein isothiocyanate
GFP Green fluorescent protein
hBMSCs Human bone marrow stromal cells
HEPES 4 -(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HUVEC Human umbilical vein endothelial cell
HVSC Human vena saphena cell
INM Inner nuclear membrane
JNK cJun N-terminal kinase
LIMAP Light Induced Molecular Adsorption of Proteins
LINC Linker of Nucleoskeleton and Cytoskeleton
LMNA Lamin A
LMNB Lamin B
MEF Mouse embryonic fibroblast
MTFM Molecular-tension-based fluorescence microscopy
NaOH Sodium hydroxide
ONM Outer nuclear membrane
PEG Polyethylene glycol
PBS Phosphate buffered saline
PLPP Photoinitiator
PDMS Polydimethylsiloxane
PLL Poly-L-lysine
RFGD Radio frequency discharge
ROCK Rho-associated kinase
SEM Standard error of the mean
SF Stress fiber
SiR Silicon rhodamine
SVA Succinimidyl valerate
TE Tissue Engineering
1
G E N E R A L I N T R O D U C T I O NGood order is the foundation of all things. -Edmund Burke
1.1 i n t r o d u c t i o n
Loss of organ function is a major cause of mortality. Organ transplantation to replace damaged or non-functioning tissues remains the only therapeutic op-tion for most pathologies leading to organ failure. Unfortunately, this opop-tion is greatly limited because of the global shortage of organs for transplantation and immunocompatibility issues [152]. The emerging fields of regenerative medicine and tissue engineering promise to solve this by using the ability to replace or regenerate damaged or diseased tissues by exploiting the body’s regenerative capacity. These fields are now focusing attention on understand-ing livunderstand-ing cells.
In vivo, adherent cells of tissues are interconnected with the surrounding extracellular matrix (ECM), a three-dimensional (3D) scaffold responsible for the structural integrity of tissues. Cells interact with their ECM over a range of length scales (nanometer to hundred of micrometers) and the ECM provides biochemical and biophysical signals instructing cells to grow, dif-ferentiate, and organize, which influence tissue development and remodel-ing. In vivo, most tissues in the human body exhibit anisotropy, or direction-dependent properties, coordinated by a specific spatial organization of cells. Cellular organization plays a crucial role in the micro-architecture of tissues. This is demonstrated by the fact that biological and mechanical functioning of most tissues is dictated by the arrangement of cells [81]. For instance, the typical basket weave structure of cardiac muscle tissue ensures efficient ejec-tion of blood from the heart [41], whereas the perfectly aligned organization of tendons warrants proper force transmission between muscle and bone [207,234]. The cellular orientation response is induced by the specific orga-nization of the intracellular structural components (Fig.1.1A).
In tissue engineering and regenerative medicine, biomaterial scaffolds are used to serve a role similar to the ECM by providing mechanical and ad-hesive support to the cells. These instructive materials must replicate the complex three-dimensional (3D) structures of the target tissues to regener-ate. Approaches in scaffold designing attempt to mimic the functionality present in the native ECM in order to control cell behavior. Despite the im-portance of cellular organization, the fundamental question of how a cell senses and responds to structural environments and organizes itself in phys-iological tissue context is still poorly understood. Therefore, to recapitulate
the anisotropic nature of native tissues, an in-depth understanding of cell be-havior under physiological conditions and in response to the environment is needed.
1.2 t h e s t r u c t u r a l i n t e r a c t i o n b e t w e e n t h e e x t r a c e l l u l a r e n v i r o n m e n t a n d t h e i n t r a c e l l u l a r c o m p o n e n t s
The natural ECM is a complex fibrous network of proteins (e.g. collagens, fibronectin, laminins), growth factors, and polyssaccharides arranged in a tissue-specific architecture. At the macro-scale, the relative amount of these molecular constituents endows the tissue with tensile strength, resilience, and resistance against compressive forces, while further fine-tuning of load-bearing properties takes place via modification of protein fiber architecture, including fiber length, diameter, and cross-linking [145]. At the micro-scale, the fiber architecture provides physical structure and a biochemical context to the cellular micro-environment. In this thesis, we focus on one of the core components of the ECM, fibronectin (FN), whose organization into fibrillar networks is driven by cells [71]. During tissue repair, FN promotes cell pro-cesses critical to tissue regeneration and regulates the deposition and organi-zation of other ECM proteins [233]. The ECM also provides anisotropic me-chanical, biochemical, and physical cues that regulate cell shape, movement, function, as well as cell fate. For example, as shown by Engler et al. [75], the stiffness of the ECM can guide stem cells into specific cell types. The mech-anisms employed by the cells to respond and adapt to these physical envi-ronmental cues is by transmitting the signals from the micro-environment to the interior of the cell via a distinct structural interconnected pathway which we define as “the structural mechanotransduction pathway”. Mechanobiol-ogy is the emerging field that focuses on the way in which cells sense and affect their mechanical environment. In Chapter 2 we will provide a de-tailed background on the structural mechanotransduction pathway. Briefly, the integrins are the first components of this pathway, physically linking the ECM (outside of the cell) with the actin cytoskeleton (inside of the cell). Integrins are transmembrane heterodimeric receptors that specifically bind to fibronectin or other ECM components. Via the integrins, large protein complexes called focal adhesions (FAs) physically connect the ECM via the cell cytoskeleton to the nucleus (see Fig. 1.1B). The cell cytoskeleton is a space-filling network of protein filaments that enables cells to maintain their
shape and mechanical strength and comprises three types of protein fila-ments: actin, microtubules and intermediate filaments [126]. In this thesis, we concentrate on the actin filaments, since these structures are directly con-nected to the focal adhesions and play an important role in determining cell organization.
Figure 1.1:A) Structural anisotropy at different length scales: from tissues to single cells to intracellular structural components. B) Schematic illustration highlighting the intracellular structural components forming the structural mechanotransduction pathway. Integrins at the plasma membrane connect the extracellular environment (substrate) to the actin cytoskeleton (green). The connection is realized, in the cellular interior, by the focal adhesions (magenta). Within the actin cytoskeleton filaments, two kinds of fibers can be distinguished. The basal actin fibers (light green) that can be found underneath the nucleus (blue) and the actin cap fibers running on top of the nucleus (dark green).
1.3 u n d e r s ta n d i n g t h e i n f l u e n c e o f a n i s o t r o p i c
e n v i r o n m e n t s o n t h e c e l l u l a r o r i e n tat i o n r e s p o n s e
Already more than 50 years ago, Weiss reported that anisotropic cellular micro-environments lead to preferential cell orientation in the direction of the substrate anisotropy, a phenomenon referred to as contact guidance [283]. As a response to the substrate anisotropy, cells reorganize their intracellular structural components (i.e. FAs, cytoskeleton, and nucleus), suggesting that these components play a fundamental role in cell functioning. Although ex-tensively studied ever since (see Chapter 2), integrating the results of these different investigations is a difficult task and the underlying mechanisms are not clear, because of the lack of a systematic approach.
To study the cellular orientation responses to substrate anisotropy and the role of the intracellular structural components, we propose to use a system-atic approach to quantify the influence of a given parameter of the micro-environment on cell alignment. This is aided by quantitative evaluation of the intracellular structural components in conjunction with the morphology of single cells (Chapter 3). This quantitative data can reliably inform compu-tational models, i) to build intuition about potential mechanisms that under-lie cellular anisotropy sensing, ii) to enable the interpretation of sometimes counter-intuitive data, and iii) to generate novel understanding of biological phenomena by suggesting hypotheses that motivate next experiments [231]. Combining experimental data and computational modeling can aid in our current understanding on cellular anisotropy sensing.
1.3.1 Manipulating substrate anisotropy
The in vivo situation of cells in the body is mimicked by 3D culture condi-tions. However, addressing specific cellular responses to particular geomet-ric cues remains difficult due to the complexity of the architecture of in vivo 3D environments. To simplify the highly complex 3D in vivo environments, micro-fabricated devices were developed. This has triggered numerous stud-ies to unravel cellular responses to the propertstud-ies of the micro-environment. These in vitro systematic approaches can aid in identifying and understand-ing cellular behavior that is governed by the anisotropic cues of the environ-ment.
It is well known that the cellular orientation response to substrate anisotropy strongly depends on cell type and the size of the anisotropic features. In this thesis, we use mammalian vascular derived cells (Chapter 3-5), i.e. Human Vena Saphena Cells (HVSCs). These cells are used in our laboratory for tis-sue engineered heart valves and vessels. Previous studies have shown that these cells can sense and respond to mechanical cues [164,249]. In Chapter 6 we use mesenchymal stem cells, a cell source which has been widely utilized in regenerative medicine due to their self-renewal capacity and potential to differentiate into distinct cell types for tissue homeostasis. To geometrically control the intracellular structural components of these cells, we developed a model set-up consisting of micro-fabricated substrates that has the ability to isolate and vary a single environmental parameter in order to decipher each components’ contribution to cellular alignment (Chapter 4 and 5). This set-up consists of patterns with features varying from the submicron to the micrometer length scale (Fig.1.2). We hypothesize that micron-sized features (around 200 µm, which is the length-scale of a mammalian cell) will mainly affect cell morphology, while submicron-sized features (around 2 µm, which is the length scale of a focal adhesion) are mainly involved in controlling intracellular structures.
Simple or complex patterns on various surfaces can be created by microfab-rication techniques. The techniques offer the possibility to control the spatial distribution of proteins on flat two-dimensional (2D) substrates at the mi-croscale. Among these techniques, a method called microcontact printing was introduced in the mid-1990s by the Whidesides group [288], which in-volves creating protein patterns using a bas-relief of the desired pattern on a silicon master created by photolithography. An elastomeric stamp made from polydimethylsiloxane (PDMS) can be cast onto the photolithographic master, cured and peeled off. This microstructured PDMS stamp is then cov-ered with a solution of protein for inking and brought into contact with the substrate to create the desired patterns upon which cells are cultured [39]. Subsequently, a coating to block nonspecific protein adsorption between the patterns is applied, to ensure that cells are only able to adhere to the patterns and not in other regions. In this thesis we use the microcontact printing tech-nique to create micron-sized patterns of fibronectin on flat PDMS substrates treated with Pluronic F127. In this way, we can restrict cell spreading to the patterns of adhesive material and manipulate cell organization (see Chapter
4 and 5).
Although the aforementioned micropatterning techniques allow the control of cell adhesion, they are also tedious, complex, non-quantitative. To cre-ate more controlled and biomimetic environments, recently, a new commer-cially available system, called PRIMO (Alvéole, Paris France) was developed. PRIMO is a contact-less and maskless UV projection system relying on the spatial control of a UV light pattern that is projected onto a substrate. This technology is based on a water-soluble photoinitiator (PLPP) that allows for tuning the antifouling properties of the substrate when exposed to the UV-light. The substrates are passivated with an anti-fouling coating consisting of polymer brushes. At the illuminated locations, the PLPP cleaves these polymer brushes, thereby creating patterns of predefined shapes and sizes. Subsequent incubation of the substrate with a protein solution results in pro-tein absorption at the illuminated regions. PRIMO allows to project any pat-terns of UV-light through the objective of a conventional inverted microscope [245]. In this way it becomes possible to design seemingly endless variations of micropatterning experiments. For example, this technique could be used to print proteins onto other types of substrates, such as curved (two-and-a-half dimensional (2.5D)) PDMS substrates or other materials.
In vivo, cells can respond to geometrical cues within a wide range of length scale; from (sub) micron meter to features larger than cell size, however, the mechanisms by which the cellular orientation response is influenced by ge-ometrical features of different scales remains poorly explored. Furthermore, while the orientation response to single geometrical cues has been exten-sively studied, very little is known about the cell response to multiple geo-metrical cues. The combination of two individually controlled cues on the cellular orientation response, would give insights on cell adaptation in the native cellular micro-environment, as also is discussed in (Chapter 2). In Chapter 6, we present a protocol using PRIMO to create contact guidance cues in the form of fibronectin lines of a few micrometers on top of cylin-ders of various diameters that mimic fiber structures that can be found in vivo as wells as in scaffold that provide curvature guidance cues. Using this approach, we aim to mimic the natural 3D micro-environment of cells more closely.
1.3.2 Understanding the cellular response to substrate anisotropy
To study cell organization, one of the primary methods to examine cells is by fluorescence microscopy. Many approaches for quantifying cellular align-ment have been successful, and powerful statistical techniques have been developed to analyze the obtained data. However, while this way of analyz-ing is fairly accurate, it suffers from certain limitations: the current manual method is susceptible to inter-operator angle measurement variability, it is time-consuming, and the method becomes highly inefficient when the data set ranges from hundreds to thousands of images. Therefore, an automated, objective, and repeatable image processing technique is needed to address these issues. In Chapter 3 we show the development of a fast, user-friendly, and automated image analysis algorithm to capture and characterize the cellular, nuclear and FA morphological changes in response to a variety of environmental changes as well as manipulations of cellular components of mechanotransductions with a high level of accuracy.
The observation of dynamic changes provides more insights into the dynam-ics of intracellular components, as compared to a snapshot provided by imag-ing studies of fixed cells. A range of techniques has been developed and used successfully for studying intracellular dynamics within an in vitro setting [292]. Live-cell fluorescence microscopy is a valuable tool which allows the observation of the intracellular structural components and cellular processes in real time and across time. The strength of live-cell fluorescence imaging is in the specificity with which intracellular structures can be labeled, imaged and analyzed. The success of live-cell imaging relies on various factors in-cluding the controlling devices for keeping a physiological environment for cells, the development, transfer and expression of candidate genes and/or fluorescent proteins in cells (i.e. transfection) and minimizing phototoxicity, while extracting data with the most spatial and temporal resolution possible [77]. Transfection of primary cells has proven to be very difficult. The recent development of the BacMam system (i.e. a modified insect cell virus as a vehicle to efficiently deliver and express proteins) [143] together with recent advances in fluorescent probes tagged in the near-infrared (IR) spectrum (i.e. silicon rhodamine (SiR)-actin) [168], make it possible to follow the dynam-ics of the actin cytoskeleton and FA in primary cells with improved spectral properties for long-term in time (Chapter 5).
By using quantitative data in conjunction with live-cell imaging of experi-ments on cell organization to individual biophysical cues, we are one step closer to improving our current understanding on cellular anisotropy sens-ing. As demonstrated by Mak et al. [171] this experimental data can also complement and strengthen computational models aiming to unveil under-lying mechanisms and bridge current gaps in our current understanding. Therefore, in this thesis we will combine our obtained quantitative exper-imental data with the results from theoretical framework in collaboration with computational scientists (Prof. Vikram Deshpande, Hamsini Suresh and Tommaso Ristori). This to increase our conceptional and quantitative insights on cellular anisotropy sensing (see Chapter 4 and 5).
Figure 1.2: Schematic representation of the experimental setup to systematically study the effects of substrate anisotropy on cell organization. For this we create adhe-sive patterns of fibronectin (red) across different length scales on non-adheadhe-sive (grey) substrates and quantitatively study the intracellular structural components by using fixated samples and live-cell imaging. Scale bar: 50 µm.
1.4 t h e s i s o u t l i n e
The anisotropy of the micro-environment plays a large role in cell organi-zation. However, it is largely unknown how cells sense en respond to
sub-strate anisotropy and we lack an in-depth understanding of the underlying mechanisms. Most of the explained mechanisms has been based on the differ-ent intracellular compondiffer-ents of the structural mechanotransduction pathway. However, these explanations are specific to experimental setups, the cell type, type of cues, and length scales of the system. Therefore, in this thesis we aim to get more insights in how cells sense and respond to anisotropic substrates by systematically controlling the intracellular structural components and by combining the obtained experimental data with computational modeling. In Chapter 2 we provide an overview of the knowledge obtained in the last decades about the structural mechanotransduction pathway. We also report the current status about the knowledge we have on the cellular orientation response induced by the application of substrate anisotropy (e.g. biochemi-cal and topography) and cyclic strain.
To uncover the mechanisms behind cellular anisotropy sensing, a robust quantitative approach to analyze the morphology of cells and the intracel-lular structural components is required. As such, we developed a fast, user-friendly, and automated image analysis algorithm of cellular, nuclear and FA morphological changes to capture and characterize these structural compo-nents with a high level of accuracy which we describe in Chapter 3.
This quantitative approach was used in combination with a numerical ap-proach to understand the mechanisms behind cellular anisotropy sensing, e.g. contact guidance. To systematically study the mechanisms behind con-tact guidance, we first created, via microconcon-tact printing, stripes of fibronectin and determined where contact guidance emerges (Chapter 4). In Chapter 5 we tried to understand the biological origin of contact guidance by mimick-ing the native-like fibrillar ECM via patternmimick-ing multiple lines of fibronectin. A layer of complexity was added to the experiment to reconstruct the na-tive 3D ECM by presenting two different cues to the cells using a new pho-topatterning system. In Chapter 6 we show how to create fibronectin lines (contact guidance) on top of curved substrates (curvature guidance) in order to understand the effects of combined cues on cell organization.
Chapter 7 provides and discusses an overview of the general findings of the presented data and discusses the future perspectives that remain.
2
H E A D I N G I N T H E R I G H T D I R E C T I O N :U N D E R S TA N D I N G C E L L U L A R O R I E N TAT I O N R E S P O N S E S T O C O M P L E X B I O P H Y S I C A L E N V I R O N M E N T S
The aim of cardiovascular regeneration is to mimic the biological and me-chanical functioning of tissues. For this it is crucial to recapitulate the in vivo cellular organization, which is the result of controlled cellular orienta-tion. Cellular orientation response stems from the interaction between the cell and its complex biophysical environment. Environmental biophysical cues are continuously detected and transduced to the nucleus through en-twined mechanotransduction pathways. Next to the biochemical cascades invoked by the mechanical stimuli, the structural mechanotransduction path-way made of focal adhesions and the actin cytoskeleton can quickly trans-duce the biophysical signals directly to the nucleus. Observations linking cellular orientation response to biophysical cues have pointed out that the anisotropy and cyclic straining of the substrate influence cellular orientation. Yet, little is known about the mechanisms governing cellular orientation re-sponses in case of cues applied separately and in combination. This chap-ter provides the state-of-the-art knowledge on the structural mechanotrans-duction pathway of adhesive cells, followed by an overview of the current understanding of cellular orientation responses to substrate anisotropy and uniaxial cyclic strain. Finally, we argue that comprehensive understanding of cellular orientation in complex biophysical environments requires systematic approaches based on the dissection of (sub)cellular responses to the individ-ual cues composing the biophysical niche.
The contents of this chapter are based on:
C. Tamiello*, A.B.C. Buskermolen*, F.P.T. Baaijens, J.L.V. Broers, C.V.C. Bouten, Head-ing in the right direction: UnderstandHead-ing cellular orientation responses to complex biophysical environments, Cellular and Molecular Bioengineering, 2015
2.1 i n t r o d u c t i o n
Cardiovascular regenerative medicine has emerged as a promising approach to replace or regenerate damaged or diseased cardiovascular tissues. This in-terdisciplinary field, at the cross-section of engineering and life sciences, has the potential to restore normal cardiovascular function by using (the proper-ties of) living cells in combination with biomaterials, genes, or drugs. Novel in-situ tissue engineering approaches rely on the regenerative potential of the body itself by guiding and controlling cell behavior inside the human body with tailored biomaterials. The premise of this approach is that, to re-capitulate tissue function, an in-depth understanding of native cell behavior under physiological conditions and in response to a biomaterial is needed. Only then, strategies for controlling cell behavior can be designed towards the restoration of tissue functionality and mechanical integrity [101].
One crucial, but often overlooked, aspect of mimicking native tissue func-tioning is obtaining and retaining cellular organization. The importance of cellular organization is demonstrated by the fact that biological and mechan-ical functioning of most tissues is dictated by the cellular arrangement [81]. The tissues of the cardiovascular system are highly organized. For instance, the myocardial wall [225], heart valves [229] and larger arteries [258] are char-acterized by a layered structure with a well-defined cellular arrangement conferring the tissues their native unique anisotropic mechanical behavior needed to perform their function. Given the correlation between structural organization and function, it becomes clear that the loss of cellular orga-nization is indicative of tissue malfunctioning, which can eventually lead to pathophysiological conditions. The disorganized arrangement of cardiac cells, for example, is a histological hallmark of cardiac dysfunction in hyper-trophic cardiomyopathy [44,110,113,190].
Cellular organization in cardiovascular tissues depends on the complex in-teractions between cells, the properties of the micro-environment and the cyclic strains resulting from the hemodynamic environment. Living adher-ent cells actively interact, respond, and adapt to biochemical and biophysi-cal perturbations. These perturbations trigger intracellular signaling events leading to specific cellular mechanoresponses capable of directing biological relevant processes such as cell differentiation, proliferation and contractility. The mechanisms employed by cells to respond and adapt to the
biochem-ical and biophysbiochem-ical cues of the micro-environment consist of a myriad of distinct but interconnected pathways whose details remain to be unraveled. The outside-in and inside-out feedback loop, referred to as mechanotrans-duction, is traditionally regarded as the process of converting mechanical stimuli into biochemical signals. Recently, it has been suggested that the structural pathway connecting the extracellular environment to the nucleus, [280] here defined as “the structural mechanotransduction pathway”, might be as important as the biochemical transduction pathway for conducting bio-physical signal to the nuclear interior. This new concept is supported by the fact that the long-range force propagation into the cell, resulting in deforma-tions deep inside the cytoskeleton and nucleus, occurs 40 times faster than biochemical signaling [182]. The structural mechanotransduction pathway consists of structural load bearing elements, such as integrins and focal adhe-sion complexes at the cellular membrane, and actin cytoskeleton stress fibers connected to the nucleus via so-called LINC (Linkers of the Nucleoskeleton and Cytoskeleton) complexes. Experimental evidence for this direct intercon-nection arises from studies where forces were applied directly to a small spot on the cell surface and consequently induced deformations and movements in the cellular interior [162,173]. Clearly, defects in the complex and delicate interplay between the cell and its micro-environment resulting, for instance, from aberrations of the structural mechanotransduction pathway, may re-sult in altered cellular mechanoresponse, in case no compensatory signaling mechanisms arise.
The recent development of micro-fabricated devices capable of effectively mimicking controlled biophysical cues has triggered numerous studies aim-ing at unravelaim-ing cellular responses to the properties of the micro-environ-ment. It has become clear that cell orientation is actively determined by the actin stress fibers [256]. Stress fiber orientation and, consequently, cellular alignment can be induced by two important biophysical cues of the cellular environment, such as those occurring during hemodynamic loading: i) the anisotropy of the environment, e.g. the substrate on which cells are cultured and ii) uniaxial cyclic strain [17,159]. These cues induce rapid and specific orientation of the intracellular elements of the structural mechanotransduc-tion pathway, i.e. the focal adhesions, the actin cytoskeleton and the nucleus, suggesting that the direct structural mechanotransduction pathway plays a fundamental role in the cellular orientation response [54,134]. Although a wealth of information has been obtained by recent in vitro
mechanotransduc-tion studies at the tissue-level, single cell observamechanotransduc-tions provide detailed mech-anistic insights towards a comprehensive understanding of cellular mechan-otransduction. Yet, integrating the results of different investigations is a dif-ficult task because of the complexity of the cellular response, which is not only highly dependent on the choice of the physical and mechanical experi-mental parameters, but also dependent on the cell-type. Moreover, the effects of combined biophysical cues on the cellular orientation response have just begun to be explored.
Here, we present a state-of–the-art review on the complex interplay between cells, topographical and cyclic strains cues of the extracellular environment, with a focus on cells of the cardiovascular system. Focusing on single cell ob-servations, we first introduce the structural mechanotransduction pathway, i.e. the connected cellular components forming the physical link between the extracellular environment and the nuclear genome. Then, we continue our discussion with a review of experimental observations regarding cellular ori-entation response to anisotropy of the substrate and uniaxial cyclic strain in 2D environments. We conclude with a brief outlook on future research direc-tions for improving our current knowledge of cellular mechanoresponse to complex biophysical environments.
2.2 t h e s t r u c t u r a l m e c h a n o t r a n s d u c t i o n pat h way: a p h y s i c a l c o n n e c t i o n b e t w e e n t h e e x t r a c e l l u l a r
e n v i r o n m e n t a n d t h e g e n o m e
In this section we provide background information on the cellular structural components forming the structural mechanotransduction pathway, i.e. the physical connection between the extracellular matrix (ECM) and the genome contained by the nucleus.
The structural components are represented by the focal adhesions situated at the cell membrane, the cytoskeletal filaments and, at last, the nucleus (Fig. 2.1). Among the cytoskeletal elements we concentrate on the actin filaments, since these structures are directly connected to the focal adhesions and play an important role in determining cell orientation [255,282]. Moreover their behavior is relatively easy to analyse and quantify from microscopy imaging as they form anisotropic networks when cells are aligned [20, 282]. In this
section also the relevance of the nucleo-cytoskeletal connections for correct mechanotransduction is elucidated.
2.2.1 Interconnection between the extracellular environment and the actin cytoskeleton
In vivo adhesive cells are embedded in a filamentous network called extra-cellular matrix (ECM). The integrins are the first components that physically link the ECM (outside of a cell) with the actin cytoskeleton (inside of the cell). Integrins are transmembrane αβ heterodimeric receptors that medi-ate cell adhesion to various ECM ligands such as collagen, fibronectin and laminin. The integrin family consists of about 25 members which are com-posed of combinations of α and β subunits, where the α subunit determines the ligand specificity for cell adhesion to the ECM [122]. During cell adhe-sion, conformational changes in the integrins are induced by bidirectional (inside-out and outside-in) signaling of mechanical and biochemical signals across the cell membrane [7,209,210]. Ligand binding to the integrins leads to clustering of integrin molecules at the cell membrane and recruitment of actin filaments inside the cell. The result of this process is the formation of the so-called nascent focal adhesion complexes (Fig.2.1A, left inset), multi-molecular complexes that consist of a large number of different proteins, including talin, vinculin, paxillin and tensin.
Focal adhesion complex formation initially starts with immature, small struc-tures (approximately 100 nm in diameter [89]). These structures reside at the leading edge in protrusions of the cells and provide the structural links be-tween the ECM and the actin cytoskeleton. Strikingly, the maturation of the small focal adhesion complexes into bigger, mature focal adhesions is de-pendent on actin cytoskeleton bundling and generation of mechanical force. The actin cytoskeleton spans the whole cytoplasm of eukaryotic cells, contin-uously remodels and reorganizes to perform specific cellular functions [178, 268]. It is made of globular actin (G-actin), which continuously polymer-izes into semi-flexible actin filaments, the filamentous actin (F-actin). F-actin assembles into bundles of fibers interconnected by actin crosslinkers (such as alpha-actinin and filamin) and motor proteins such as myosin II [191]. These bundles of F-actin fibers are referred to as stress fibers. The presence of myosin II within the stress fibers is responsible for their contractility. The
newly formed focal adhesions (FAs) reside in both central and peripheral regions of the cell. During this process the morphology of the FAs changes from a dot-like structure to a bigger and more elongated structure (2-10 um) [42,88]. This happens also as a consequence of the recruitment at the adhe-sion complex of several other proteins, for instance zyxin and alpha-actinin [302]. A critical molecule for both maturation of FAs and mechanosensing is focal adhesion kinase (FAK). This molecule is involved in the transmission of external signals to the cytoskeleton by phosphorylation [99].
The maturation of FAs provides stable adhesive interconnections between the stress fibers and the ECM. This allows the cell to probe its complex bio-physical environment in various directions and over large temporal and spa-tial scales [232]. FAs do not actively generate forces, but rather serve to reg-ulate force transmission between the cytoskeleton and ECM [192] The actin cytoskeleton is the intracellular structure able to impose increasing forces when facing growing resistance. This confers the actin cytoskeleton intrin-sic mechanosensing and ability to adapt to developing mechanical cues of the cellular environment. However, to which extent stress fibers participate in sensing and transducing environmental signals has not been fully eluci-dated yet.
2.2.2 Interconnection between the actin cytoskeleton and the nucleus
In the surrounding of the nucleus, a subset of actin stress fibers have been found to organize in thick parallel and well-ordered bundles of fibers, phys-ically anchored to the apical surface of the nucleus [137, 138]. Wirtz and co-workers have made an effort to characterize these fibers (actin cap) which are strikingly terminated by wide, long and dynamic FAs (Fig.2.1A) [28,117, 137]. First, they have demonstrated that the actin cap stress fibers differ from the conventional stress fibers found below the nucleus (basal actin layer, Fig. 2.1B). By containing more myosin II and the actin bounding protein alpha-actinin, actin cap stress fibers are very contractile and highly dynamic [183]. Furthermore, these fibers not only play a major role in shaping and position-ing the nucleus [34,117, 137, 139, 183, 267], but they are also involved in mechanosensing of substrate elasticity. For instance, cells without an actin cap were observed to be less responsive to changes in matrix elasticity. Fi-nally, fast mechanotransduction also seems to be enabled by this subset of
stress fibers. In their study, Chambliss et al. [33] showed that, in response to shear stress stimulation, cells without the actin cap build up thick stress fibers in a shorter time span as compared to in response to biochemical stim-ulation. From these findings it has become clear that the perinuclear actin cap is a key component of the physical pathway from the ECM to the nu-clear interior for mechanosensing and mechanotransduction.
The coupling between the perinuclear actin cap and the nucleus (nucleo-cy-toskeletal connection) is mediated by a group of recently discovered proteins, referred to as the LINC complex (Linker of Nucleoskeleton and Cytoskeleton, Fig.2.1A right inset) [48,199,243]. Hooking at the cytoplasmic side of the nu-cleus, on the outer nuclear membrane (ONM), we find the nesprins (KASH domains proteins), which are connected to the various cytoskeletal filaments [239, 307]. Among the four variants of nesprins, nesprin-1 and -2 bind to actin filaments [104]. Nesprins, in turn, bind to SUN domain proteins span-ning the whole nuclear envelope reaching the nuclear interior. SUN proteins then bind to lamins, a family of type V intermediate filaments underlying the inner nuclear membrane (INM) [108]. Lamins in turn physically connect to chromatin. Thus, in this way a physical bridge is formed from the cellular exterior via focal adhesion complexes, actin, the LINC complex, and lamins to chromatin.
Lamins form an elastic meshwork called nuclear lamina (Fig.2.1a, right in-set)[52,200]. Lamins consist of two main subtypes, A- and B-type lamins (en-coded by the gene LMNA, or LMNB1 and LMNB2 respectively)[103]. While B-type lamins are essential for cell survival, A-type lamins are thought to contribute significantly to the maintenance of mechanical integrity of the nu-cleus [24,115,150,263]. The nuclear lamina interacts also with the chromatin of the nucleoplasm, and therefore plays a major role in gene expression, DNA replication and repair, chromatin organization and transcriptional re-sponse [58,98,236,309].
The role of the nucleo-cytoskeletal connection in force transmission has been examined recently by many groups. The results of various experimental ap-proaches based on two- and three-dimensional substrates or application of mechanical load, have shown that the structural integrity of this connection is indeed needed for propagation of forces to the nucleus. Indirect demon-stration has come from studies employing LMNA-depleted cells. By using
this model, it has been shown that nuclear deformations in response to local cellular membrane stretch are completely abolished [162]. In addition, the studies by Poh et al.[205] and Zwerger et al. [310] have provided direct evi-dence that forces are not transmitted to the nucleus when LMNA is depleted from cells, thus when the nucleo-cytoskeletal connection is lost. Recently, it has emerged as well that the tension exerted by the actin on the nucleus directly mediates the spatial polarization of nuclear lamina and the intranu-clear architecture [141]. In cells lacking A-type lamins, the formation of a nuclear actin cap is partially abolished [137]. Also, the impaired activation of mechanosensitive genes has been reported in studies with cells lacking A-type lamins [115,149].
A number of other studies in which either the LINC complex was disrupted or a loss of lamins was induced, support these findings adding that also other cellular functions such as migration, polarization and developmental processes become affected [25,155]. Although the role of the LINC complex in force propagation to the nucleus has been clarified, controversy remains about its impact on the activation of mechanotransduction pathways. Clues to understand these mechanisms might come from studying diseases arsing form mutations in any of the components connecting actin to the nucleus.
Figure 2.1:The structural mechanotransduction pathway and cellular orientation re-sponse to anisotropy of the substrate and uniaxial cyclic strain. A) Schematic illus-tration highlighting the (protein) structural elements forming the structural mechan-otransduction pathway. Integrins at the plasma membrane connect the extracellular environment (substrate) to the actin cytoskeleton. The connection is realized, in the cellular interior, by the focal adhesion complex (magenta). Within the actin cytoskele-ton filaments, two kinds of fibers can be distinguished. The basal actin fibers (light green) that can be found underneath the nucleus and the actin cap fibers (dark green) running on top of the nucleus (blue). Actin cap fibers are connected to the nuclear in-terior via the LINC complex and lamins, a group of proteins underlying the nuclear membrane. This network of components forms a direct connection between the extra-cellular environment and the nuclear interior and function as a fast passing system for the biophysical stimuli. B) Schematic illustration of cellular response to substrate anisotropy and uniaxial cyclic strain. When plated on an anisotropic substrate (left), the cell tends to align in the direction of the anisotropy. Focal adhesions as well as the actin cytoskeleton align accordingly. The side view shows the arrangements of the actin cap and basal actin fibers. Upon uniaxial cyclic strain (right), the cell responds by strain avoidance. The focal adhesions and the actin cytoskeleton align at an an-gle with respect to the straining direction. Overall cell orientation coincides with the actin cytoskeleton orientation. Note that the focal adhesions associated with the actin
2.3 c e l l u l a r o r i e n tat i o n r e s p o n s e t o s u b s t r at e a n i s o t r o p y a n d c y c l i c s t r a i n
In the previous section, in order to appreciate the inside-in part of the cellu-lar mechanotransduction, i.e., how environmental signals are transmitted to the nucleus, we introduced the components of the structural mechanotrans-duction pathway interconnecting the extracellular environment and the nu-cleus. To get a comprehensive understanding of the interplay between cel-lular responses and complex biophysical environments, it is also necessary to have a deep understanding of the inside-out signaling used for cellular mechanoresponse, i.e., how cells respond to environmental cues and which mechanisms are employed by cells for mechanoresponse. In this section we discuss the cellular orientation response to substrate anisotropy and uniaxial cyclic strain (Fig. 2.1B), focusing on the main components of the structural mechanotransduction pathway, i.e. the focal adhesions (FAs), the actin cy-toskeleton and the nucleus.
2.3.1 Cellular orientation response to substrate anisotropy
Various biophysical cues such as topography, cyclic strain and the mechani-cal properties of the extracellular environment can induce the alignment of adherent cells by promoting an anisotropic arrangement of structural com-ponents at the subcellular level. In 1912, Harrison reported for the first time that the topography of a substrate could influence cell behavior [109]. Weiss confirmed this in 1945, with the observation that cells preferentially orient and migrate along fibers, an organization principle he named contact guid-ance [283]. Today the connotation of this term is slightly different. Contact guidance is now regarded as the ability of cells to sense and align with the anisotropy of the surrounding micro-environment. Recent developments in microfabrication technologies have led to the manufacturing and application of a variety of substrates with different geometries and length scales, from which several substrates can be used to study contact guidance. Observations obtained using microfabricated substrates engineered to induce contact guid-ance, have confirmed that a variety of tissue cells, ranging from endothelial cells [67,142,260], to fibroblasts [60,69,193,269,270], and smooth muscle cells [223] orient along the direction of the anisotropy of the substrate. A summary of illustrative studies showing the response of cells of the
cardio-vascular system to anisotropic features of the culture substrate in the sub-micrometer to sub-micrometer scale is reported in Table2.1.
The most used substrates for studying contact guidance are microgrooves, i.e. microengineered arrays of parallel micrometer-sized grooves and ridges. When culturing adherent cells on these substrates it is observed that, at the subcellular level, the FAs and actin fibers follow cellular orientation (Fig. 2.1B, left). However, the specific response of these structural cellular compo-nents depends on many parameters such as groove width [67,69,142,226, 270], ridge width [23,60,67,69,142,252], groove height [23,46,252,260,270] and surface treatment [67]. The general trend is that when either the groove width or groove height increases, the cell forms FAs on the ridges and con-sequently orients in their direction. Next to these observations, several the-oretical frameworks have been elaborated for explaining cell alignment in relation to the microgroove’s parameters. The schematic representation of these theories is shown in Fig.2.2.
• The mechanical restriction theory by Dunn and Heath focuses on the relative inflexibility of cytoskeletal structures as a primary regulator of cellular alignment [70]. The shape of the substratum is demonstrated to impose mechanical restrictions for the formation of cytoskeletal protru-sions, called filopodia, as recently shown also by Zimerman et al.[308] and Ventre et al.[262]. According to this theory, the distance between the anisotropic features, either the groove width (Fig. 2.2B, left), on microgrooved substrates or the distance between adhesive lines on flat substrates, is the crucial factor for cell alignment. If this distance cannot be bridged by the formation of any filopodia, cells become highly po-larized and elongate in the direction of the substrate anisotropy. When cells align because of this mechanism, actin filaments as well as long FAs are observed in the direction of the anisotropy. These FAs are usu-ally anchored to thick stress fibres and, therefore, are presumably the FAs of the actin cap stress fibers.
• The focal adhesion theory by Ohara and Buck proposes that the ori-entation of cells is caused by the tendency of FAs to maximize their contact area [196]. According to this theory, on a microgrooved sub-strate, FA maturation and, consequently, cell alignment occur along the ridge only if ridge width (Fig. 2.2B, center) is comparable to the size of a FA. An argument against this theory is the observation of
focal adhesions oriented both perpendicular and parallel to ridges of the microgrooved substrate [193,262,308]. However, as pointed out by Ventre et al.[262], FAs observed perpendicular to the ridge direction are unstable and connected to isolated actin fibers, while the FAs parallel to the ridge are mature and connected to stress fibers. This ultimately guides cellular orientation in the ridge direction.
• Discontinuity theory: more recently Curtis and Clark proposed the idea that sharp discontinuities in the substrates, e.g. edges of micro-grooves, induce cell alignment by triggering, first, actin condensations in these locations and, consequently, promoting focal adhesion forma-tion at the same place [49]. Despite the fact that this theory includes both the involvement of FAs and actin filaments in cell alignment, it raises the question of how cells sense discontinuity, as already dis-cussed by Curtis et al.[49]. Clark et al.[46] observed that by increasing the grooves height (Fig. 2.2B, right), more cells orient in the direction of the microgrooves. Based on these observations, it is proposed that for sufficiently high microgrooves, cells are more exposed to substrate discontinuity and, as a result, align along the microgrooves.
Figure 2.2:Cellular orientation response to microgrooves. A) Schematic illustration showing the overall cellular orientation response from cell adhesion to alignment on a microgrooved substrate. At the moment the cell adheres to the microgrooved substrate, the cell undergoes spreading followed by cell alignment, i.e. orientation along the direction of the microgrooves, a phenomenon called contact guidance. The parameters characterizing the microgrooved substrate are pointed out with light blue arrows: groove width, ridge width and groove height. B) Schematic represen-tation of the proposed mechanisms explaining contact guidance in relation to the mi-crogroove’s parameters. (Top) No cell alignment and (bottom) cell alignment. (Left) groove width – mechanical restriction theory. When the microgrooves are too nar-row, cells’ filopodia succeed in bridging the space between two consecutives ridges. Therefore, the cell does not align (top). When the width of the microgrooves increases, filopodia are not able to bridge two consecutive ridges, giving the signal for cell align-ment in the direction of the microgrooves (bottom). (Center) ridge width - focal adhe-sion theory. Ridge width influences the orientation and maturation of focal adheadhe-sions. Wide ridges do not impose geometrical confinement on the focal adhesion (magenta). Therefore, the maturation of the focal adhesions can occur in both directions, pre-venting any cell alignment (top). Narrower ridges impose geometrical confinement to the focal adhesions, which tend to maximize their contact area with the substrate. As a result, focal adhesions align and mature in the direction of the ridges, i.e. the direction of the microgrooves (bottom). (Right) groove height – discontinuity theory. For low microgrooves, the cell sinks into the microgrooves and, consequently, it does not align in direction of the microgrooves (top). For sufficiently high microgrooves, the cell senses the discontinuities of the microgrooves represented by their edges
Figure 2.3: Cellular orien-tation response to a two-dimensional anisotropic
environment.
Repre-sentative microscopy image of a myofibroblast
(Human Vena Saphena
Cell) cultured on top of microcontact printed fibronectin (red) lines (10 µm width and 10 µm inter-line spacing) on polydimethylsilox-ane (PDMS). The focal adhesions are shown in magenta, the actin stress fibers in green, and the nucleus in blue. The cell orients indirection of the lines. The focal adhesions and the actin stress fibers follow cellular orientation. Although these theories have
shed light on the possible mechanisms behind contact guidance, the role of each individual structural compo-nent has not been fully elu-cidated yet. A straightfor-ward approach to investi-gate the influence of the actin cytoskeleton in cellular alignment to microgrooves consists by inhibiting the actin cytoskeleton via dis-rupting agents, such as per-formed by Walboomers et al. [269] and Gerecht et al. [90]. On one hand, Walboomers et al. observed that fibrob-lasts can still align along the microgrooves even if the polymerization of actin is inhibited with the use of
cytochalasin-B [269]. Contrarily, Gerecht et al. found that by adding actin disrupting agents to human embryo stem cells on sub-micrometer sized grooves, the morphology of the cells gets rounder [90]. These results illus-trate that there is no consensus yet on the role played by the actin cytoskele-ton in the cellular response to contact guidance.
To unravel the relevance of each of the cellular components in the con-tact guidance phenomenon, a systematic approach is, in our view, needed. The various substrate features creating anisotropy need to be dissected (e.g. height, edges, biochemical patterning) and it is necessary to distinguish be-tween substrate anisotropy by biochemical features (e.g. geometrical fea-tures given by printing of extracellular matrix proteins), i.e. a purely two-dimensional (2D) environment, and substrate anisotropy by topographical features (e.g. pillars, posts, microgrooves, fibers), here named two-and-a-half-dimensional (2.5D) environment. The first step towards this systematic approach, is neglecting the influence of the height of topographic features
(e.g. discontinuity). Thus, as a first step, it is suggested to study contact guidance in 2D environments. Pure biochemical anisotropic features can be produced for instance by microcontact printing. According to this methodol-ogy, an elastomeric stamp of polydimethylsiloxane (PDMS) incubated with an extracellular matrix protein (e.g. fibronectin) can be used to create adhe-sive patterns on flat surfaces, such as glass or PDMS. The bare regions are then backfilled with a non-adhesive protein or polymer, to avoid non-specific cell adhesion. Microcontact printing has proven to be a useful technique to adhere cells to single or multiple islands [39, 40]. In this way one can ge-ometrically control cell adhesion to regulate cell functions. However, there are only limited studies where this technique has been used to induce cellu-lar alignment via printed lines whose width is in the order of micrometers [5, 290, 308]. In our view, these kinds of studies will elucidate the precise mechanisms behind cellular alignment (Fig.2.3).
T able 2 .1 : Experimental inv estigations on cell orientation response induced b y anisotr op y of the substrate. Cell type Method Parameters Main obser v ed results S our ce Fibroblasts (REF 52 cells) Parallel micr ocontact printed fi-br onectin lines Lines: 2 µm wide seperated b y 4, 6, 10 or 12 µm wide stripes FAs ar e for med at adhesiv e lines [ 308 ] Glass If spacing is lar ger than 6 µm, FAs orient either per pendicular to the lines or in the dir ection of the lines. Actin str ess fibr es can cr oss se v eral stripes when the non-adhesiv e spacing is smaller than 6 µm. W ith wider spacing str ess fibr es for m betw een adjacent adhesiv e stripes or along single stripes. Chick heart fibroblasts Parallel gr oo v es Ridge width fr om 1. 65 to 8. 96 µm Focal adhesions obser v ed on the floor of the gr oo v es and at the ridges. [ 69 ] Quartz Gr oo v e width fr om 3 to 32 µm Actin bundles associated with focal contact on the floor of gr oo v es ar e parallel to the gr oo v e axis and har dly ev er nearly per pendicular to this axis. No such restriction is found on the ridges. Constant depth: 0. 69 µm Human gingiv al fibroblasts Parallel gr oo v es Ridge and gr oo v e width of 15 µm Micr otubules located at the bottom of the gr oo v es ar e the first com-ponent to align along the gr oo v es. Subsequently , focal adhesions and actin micr ofilaments align. [ 193 ] Silicon Constant depth: 3 µm At a single gr oo v e or ridge, focal adhesions ar e oriented both parallel and per pendicular to the gr oo v e dir ection T itanium coating Rat der mal fibroblasts Parallel gr oo v es Ridge and gr oo v e width fr om 1 to 10 µm Cells at surfaces with a ridge width smaller than 10 µm elongate along the surface gr oo v es. If the ridge width is lar ger than 4 µm, cellular orientation w as random and the shape of the cells became mor e cir cular . [ 60 ] PDMS Depth: 0. 45 or 1 µm The ridge width is the most important parameter , since v ar ying the gr oo v e width and gr oo v e depth does not af fect cell size, shape, nor the angle of cellular orientation. RFGD tr eatment Ridge and gr oo v e width fr om 1 to 20 µm The cells alw ays elongate in the gr oo v e dir ection without any signifi-cant dif fer ence in beha viour betw een 2– 20 µm wide gr oo v es. Ho w ev er , gr oo v e depth af fects the cellular orientation. [ 270 ] Depth: 0. 5, 1, 1. 5, 1. 8, 5. 4 µm Ho w ev er , gr oo v e depth af fects the cellular orientation. My ofibroblasts (HVSCs) Elastomeric micr oposts PDMS Elliptical micr oposts: a= 1. 5 µm, b= 0. 87 µm Elliptical micr oposts: orientation in the dir ection of the major axis of the ellipse, ev en for v er y stif f micr oposts. [ 249 ] Micr ocontact printed Human Plasma Fibr onectin on top of micr oposts Cir cular micr oposts: d = 2 µm T opographical cues induce cellular alignment. V ascular smooth muscle cells Parallel gr oo v es Ridge width: 12 µm For all gr oo v e widths inv estigated, cells align in the dir ection of the micr ogr oo v es. PDMS Gr oo v e width: 20 , 50 and 80 µm The actin filaments ar e highly aligned and parallel to the gr oo v es on the smallest gr oo v e widths. [ 226 ]
