Control Cell Alignment Research Articles

Host bone microstructure for enhanced resistance to bacterial infections

Postoperative bacterial infection is a serious complication of orthopedic surgery. Not only infections that develop in the first few weeks after surgery but also late infections that develop years after surgery are serious problems. However, the relationship between host bone and infection activation has not yet been explored. Here, we report a novel association between host bone collagen/apatite microstructure and bacterial infection. The bone-mimetic-oriented micro-organized matrix structure was obtained by prolonged controlled cell alignment using a grooved-structured biomedical titanium alloy. Surprisingly, we have discovered that highly aligned osteoblasts have a potent inhibitory effect on Escherichia coli adhesion. Additionally, the oriented collagen/apatite micro-organization of the bone matrix showed excellent antibacterial resistance against Escherichia coli. The proposed mechanism for realizing the antimicrobial activity of the micro-organized bone matrix is by the controlled secretion of the antimicrobial peptides, including β-defensin 2 and β-defensin 3, from the highly aligned osteoblasts. Our findings contribute to the development of anti-infective strategies for orthopedic surgeries. The recovery of the intrinsically ordered bone matrix organization provides superior antibacterial resistance after surgery.

Strain Gradient Programming in 3D Fibrous Hydrogels to Direct Graded Cell Alignment.

Biological tissues experience various stretch gradients which act as mechanical signaling from the extracellular environment to cells. These mechanical stimuli are sensed by cells, triggering essential signaling cascades regulating cell migration, differentiation, and tissue remodeling. In most previous studies, a simple, uniform stretch to 2D elastic substrates has been applied to analyze the response of living cells. However, induction of nonuniform strains in controlled gradients, particularly in biomimetic 3D hydrogels, has proven challenging. In this study, 3D fibrin hydrogels of manipulated geometry are stretched by a silicone carrier to impose programmable strain gradients along different chosen axes. The resulting strain gradients are analyzed and compared to finite element simulations. Experimentally, the programmed strain gradients result in similar gradient patterns in fiber alignment within the gels. Additionally, temporal changes in the orientation of fibroblast cells embedded in the stretched fibrin gels correlate to the strain and fiber alignment gradients. The experimental and simulation data demonstrate the ability to custom-design mechanical gradients in 3D biological hydrogels and to control cell alignment patterns. It provides a new technology for mechanobiology and tissue engineering studies.

Controlled Cell Alignment Using Two-Photon Direct Laser Writing-Patterned Hydrogels in 2D and 3D.

Direct laser writing (DLW) via two-photon polymerization is an emerging highly precise technique for the fabrication of intricate cellular scaffolds. Despite recent progress in using two-photon-polymerized scaffolds to probe fundamental cell behaviors, new methods to direct and modulate microscale cell alignment and selective cell adhesion using two-photon-polymerized microstructures are of keen interest. Here, a DLW-fabricated 2D and 3D hydrogel microstructures, with alternating soft and stiff regions, for precisely controlled cell alignment are reported. The use of both cell-adhesive and cell-repellent hydrogels allows selective adhesion and alignment of human mesenchymal stem cells within the printed structure. Importantly, DLW patterning enables cell alignment on flat surfaces as well as irregular and curved 3D microstructures, which are otherwise challenging to pattern with cells.

Programmable Strain Gradients in 3D Hydrogels for Guiding Complex Tissue Architecture

Biological tissues experience various stretch gradients in the body which act as mechanical signaling from the extra-cellular environment to cells. These mechanical stimuli are transferred to cell sensor proteins, triggering essential signaling cascades regulating cell migration, differentiation and tissue remodeling. Previous studies have successfully applied simple, uniform stretch to 2D elastic substrates in order to analyze the response of living cells. However, the ability to induce non-uniform strains in controlled gradients, particularly in 3D hydrogels, has proven challenging.In this study, we manipulated the geometry of 3D fibrin hydrogels embedded in punctured silicone rubber strips. The strips were then stretched using a dedicated stretching device [1,2] and imaged in real-time under confocal microscopy. The resulting strains and internal fiber alignment gradients were analyzed and compared to finite element simulations. Strain gradient control through geometric manipulation and external stretch was confirmed with a linear relationship between strain magnitude and fiber alignment in the stretch direction. Additionally, fibroblast cells embedded in the fibrin gels were found to align along the stretch direction in a gradient manner that correlate to the developed strain gradients. The experimental and simulation data confirm our ability to custom design mechanical gradients in 3D hydrogels and control cell alignment, offering a framework for further elucidation of cell response to mechanically induced signaling and providing a platform for programming cellular behavior and differentiation. [1] A. Roitblat Riba, S. Natan, A. Kolel, H. Rushkin, O. Tchaicheeyan, A. Lesman. Straining 3D hydrogels with uniform z-axis strains while enabling live microscopy imaging. Annals of Biomedical Engineering (2019). https://doi.org/10.1007/s10439-019-02426-7. [2] A. Kolel, A. Roitblat Riba, S. Natan, O. Tchaicheeyan, E. Saias, A. Lesman. Controlled Strain of 3D Hydrogels under Live Microscopy Imaging. Journal of Visualized Expirements. (Pending Publication). e61671. In-press (2020).

Use of Elastic, Porous, and Ultrathin Co‐Culture Membranes to Control the Endothelial Barrier Function via Cell Alignment

AbstractPorous membranes used in co‐culture enable the in vitro partitioning of cellular microenvironments, while still permitting physical and biochemical crosstalk between cells. Thus, features of the co‐culture membrane are crucial for recapitulating the physiological functions of co‐cultured cells. This study presents elastic, porous, and ultrathin membranes (EPUMs), which enhance cell–cell interactions and control cell alignment with surface topology created by stretching the membranes. The EPUM is fabricated using poly(lactide‐co‐caprolactone) as the base material, and the porous feature is endowed by a vapor‐induced phase separation process induced by the presence of hygroscopic salt. Owing to its elastic property, the membrane can be stretched, and the deformed porous structures on the membrane surfaces act as nanostructured topographical cues, resulting in cell alignment. By co‐culturing human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) on the opposite sides of the membrane, rapid endothelialization occurs through the membranes, as compared to the commercial membranes. Furthermore, the stretched membranes induce the alignment of hMSCs and HUVECs and ultimately exhibit enhanced endothelial barrier function. The co‐culture membrane developed in this study may provide an effective tool for recapitulating endothelial basement membranes with a controllable endothelial barrier function.

Micropatterning Decellularized ECM as a Bioactive Surface to Guide Cell Alignment, Proliferation, and Migration.

Bioactive surfaces and materials have displayed great potential in a variety of tissue engineering applications but often struggle to completely emulate complex bodily systems. The extracellular matrix (ECM) is a crucial, bioactive component in all tissues and has recently been identified as a potential solution to be utilized in combination with biomaterials. In tissue engineering, the ECM can be utilized in a variety of applications by employing the biochemical and biomechanical cues that are crucial to regenerative processes. However, viable solutions for maintaining the dimensionality, spatial orientation, and protein composition of a naturally cell-secreted ECM remain challenging in tissue engineering. Therefore, this work used soft lithography to create micropatterned polydimethylsiloxane (PDMS) substrates of a three-dimensional nature to control cell adhesion and alignment. Cells aligned on the micropatterned PDMS, secreted and assembled an ECM, and were decellularized to produce an aligned matrix biomaterial. The cells seeded onto the decellularized, patterned ECM showed a high degree of alignment and migration along the patterns compared to controls. This work begins to lay the groundwork for elucidating the immense potential of a natural, cell-secreted ECM for directing cell function and offers further guidance for the incorporation of natural, bioactive components for emerging tissue engineering technologies.

Development of orthophosphosilicate glass/poly(lactic acid) composite anisotropic scaffolds for simultaneous reconstruction of bone quality and quantity.

Reconstruction of organ‐specific architecture is necessary to recover the original organ function. The anisotropic structure of bone tissue is strongly related to the collagen fibril alignment and bone apatite crystal direction. Bone regeneration indicates following two main process; first, restoration of bone mineral density (BMD; bone quantity), and second, restoring bone apatite c‐axis orientation (bone quality). In addition to BMD, bone quality is the most important factor among bone mechanical properties. Recovery of the original bone function requires development of novel scaffolds with simultaneous reconstruction of bone quality and quantity. Herein, novel orthophosphosilicate glass (PSG)/poly(lactic acid) composite anisotropic scaffolds were developed to control cell alignment and enhance bone formation, which are important for the simultaneous reconstruction of bone quality and quantity. The strategy to control cell alignment and bone formation involved designing anisotropic scaffolds in combination with the release of therapeutic ions by PSGs. The morphology of fibrous scaffolds containing PSGs was quantitatively designed using electrospinning. This successfully modulated cell alignment and subsequent bone apatite c‐axis orientation along the fiber‐oriented direction. The released silicate and Mg2+ ions from PSGs in scaffolds improved cell adhesion, proliferation, and calcification. To best of our knowledge, this is the first report demonstrating that the anisotropic scaffolds containing bioactive glasses regenerate bone tissues with simultaneous reconstruction of bone quality and quantity via stimulating osteoblasts by inorganic ions and designing morphology of scaffolds.

Engineering macroscale cell alignment through coordinated toolpath design using support-assisted 3D bioprinting.

Aligned cells provide direction-dependent mechanical properties that influence biological and mechanical function in native tissues. Alignment techniques such as casting and uniaxial stretching cannot fully replicate the complex fibre orientation of native tissue such as the heart. In this study, bioprinting is used to direct the orientation of cell alignment. A 0°-90° grid structure was printed to assess the robustness of the support-assisted bioprinting technique. The variation in the angles of the grid pattern is designed to mimic the differences in fibril orientation of native tissues, where angles of cell alignment vary across the different layers. Through bioprinting of a cell-hydrogel mixture, C2C12 cells displayed directed alignment along the longitudinal axis of printed struts. Cell alignment is induced through firstly establishing structurally stable constructs (i.e. distinct 0°-90° structures) and secondly, allowing cells to dynamically remodel the bioprinted construct. Herein reports a method of inducing a macroscale level of controlled cell alignment with angle variation. This was not achievable both in terms of methods (i.e. conventional alignment techniques such as stretching and electrical stimulation) and magnitude (i.e. hydrogel features with less than 100 µm features).

Controlled Dynamics of Neural Tumor Cells by Templated Liquid Crystalline Polymer Networks.

The ability to control the alignment and organization of cell populations has great potential for tissue engineering and regenerative medicine. A variety of approaches such as nano/microtopographical patterning, mechanical loading, and nanocomposite synthesis have been developed to engineer scaffolds able to control cellular properties and behaviors. In this work, a patterned liquid crystal polymer network (LCN) film is synthesized by using a nematic liquid crystal template in which the molecular orientations are predesigned by photopatterning technique. Various configurations of polymer networks such as linear and circular patterns are created. When neural tumor cells are plated onto the templated LCN films, the cell alignment, migration, and proliferation are directed in both linear and curvilinear fashions following the pattern of the aligned polymer chains. A complex LCN pattern with zigzag geometry is also fabricated and found to be capable of controlling cell alignment and collective cellular organization. The demonstrated control of cell dynamics and organization by LCN films with various molecular alignments opens new opportunities to design scaffolds to control cultured cell organization in a manner resembling that found in tissues and to develop novel advanced materials for nerve repair, tissue engineering, and regenerative medicine applications.

Primary cilia control cell alignment and patterning in bone development via ceramide-PKC\u03b6-\u03b2-catenin signaling

Intraflagellar transport (IFT) proteins are essential for cilia assembly and function. IFT protein mutations lead to ciliopathies, which manifest as variable skeletal abnormalities. However, how IFT proteins regulate cell alignment during bone development is unknown. Here, we show that the deletion of IFT20 in osteoblast lineage using Osterix-Cre and inducible type I Collagen-CreERT cause a compromised cell alignment and a reduced bone mass. This finding was validated by the disorganized collagen fibrils and decreased bone strength and stiffness in IFT20-deficient femurs. IFT20 maintains cilia and cell alignment in osteoblasts, as the concentric organization of three-dimensional spheroids was disrupted by IFT20 deletion. Mechanistically, IFT20 interacts with the ceramide-PKCζ complex to promote PKCζ phosphorylation in cilia and induce the apical localization of β-catenin in osteoblasts, both of which were disrupted in the absence of IFT20. These results reveal that IFT20 regulates polarity and cell alignment via ceramide-pPKCζ-β-catenin signaling during bone development.

Bone apatite anisotropic structure control via designing fibrous scaffolds.

Bone tissue has an anisotropic structure, associated with the collagen fibrils' orientation and the c-axis direction of the bone apatite crystal. The bone regeneration process comprises two main phases: bone mineral density restoration (bone quantity), and subsequent recovery of bone apatite c-axis orientation (bone quality). Bone quality is the determinant factor for mechanical properties of bone. Control of osteoblast alignment is one of the strategies for reconstructing bone quality since the collagen/apatite matrix orientation in calcified tissues is dependent on the osteoblast orientation. In this work, fibrous scaffolds designed for reconstruction of bone quality via cell alignment control was investigated. The fibrous scaffolds were fabricated using the electrospinning method with poly(lactic acid) at various fiber collecting speeds. The degree of fiber alignment in the prepared fibrous scaffolds increased with increasing fiber collecting speed, indicating that the fibers were oriented in a single direction. The alignment of osteoblasts on the fibrous scaffolds as well as the subsequent apatite c-axis orientation increased with increasing fiber collecting speed. We successfully controlled cell alignment and apatite c-axis orientation using the designed morphology of fibrous scaffolds. To the best of our knowledge, this is the first report demonstrating that adjusting the degree of fiber orientation for fibrous scaffolds can manipulate the regeneration of bone quality.

Shape-Memory Nanofiber Meshes with Programmable Cell Orientation

In this work we report the rational design of temperature-responsive nanofiber meshes with shape-memory properties. Meshes were fabricated by electrospinning poly(ε-caprolactone) (PCL)-based polyurethane with varying ratios of soft (PCL diol) and hard [hexamethylene diisocyanate (HDI)/1,4-butanediol (BD)] segments. By altering the PCL diol:HDI:BD molar ratio both shape-memory properties and mechanical properties could be readily turned and modulated. Though mechanical properties improved by increasing the hard to soft segment ratio, optimal shape-memory properties were obtained using a PCL/HDI/BD molar ratio of 1:4:3. Microscopically, the original nanofibrous structure could be deformed into and maintained in a temporary shape and later recover its original structure upon reheating. Even when deformed by 400%, a recovery rate of >89% was observed. Implementation of these shape memory nanofiber meshes as cell culture platforms revealed the unique ability to alter human mesenchymal stem cell alignment and orientation. Due to their biocompatible nature, temperature-responsivity, and ability to control cell alignment, we believe that these meshes may demonstrate great promise as biomedical applications.

Development of bifunctional oriented bioactive glass/poly(lactic acid) composite scaffolds to control osteoblast alignment and proliferation.

During the bone regeneration process, the anisotropic microstructure of bone tissue (bone quality) recovers much later than bone mass (bone quantity), resulting in severe mechanical dysfunction in the bone. Hence, restoration of bone microstructure in parallel with bone mass is necessary for ideal bone tissue regeneration; for this, development of advanced bifunctional biomaterials, which control both the quality and quantity in regenerated bone, is required. We developed novel oriented bioactive glass/poly(lactic acid) composite scaffolds by introducing an effective methodology for controlling cell alignment and proliferation, which play important roles for achieving bone anisotropy and bone mass, respectively. Our strategy is to manipulate the cell alignment and proliferation by the morphological control of the scaffolds in combination with controlled ion release from bioactive glasses. We quantitatively controlled the morphology of fibermats containing bioactive glasses by electrospinning, which successfully induced cell alignment along the fibermats. Also, the substitution of CaO in Bioglass®(45S5) with MgO and SrO improved osteoblast proliferation, indicating that dissolved Mg2+ and Sr2+ ions promoted cell adhesion and proliferation. Our results indicate that the fibermats developed in this work are candidates for the scaffolds to bone tissue regeneration that enable recovery of both bone quality and bone quantity. © 2019 The Authors. journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1031–1041, 2019.

Liquid Crystal-Induced Myoblast Alignment.

The ability to control cell alignment represents a fundamental requirement toward the production of tissue in vitro but also to create biohybrid materials presenting the functional properties of human organs. However, cell cultures on standard commercial supports do not provide a selective control on the cell organization morphology, and different techniques, such as the use of patterned or stimulated substrates, are developed to induce cellular alignment. In this work, a new approach toward in vitro muscular tissue morphogenesis is presented exploiting liquid crystalline networks. By using smooth polymeric films with planar homogeneous alignment, a certain degree of cellular order is observed in myoblast cultures with direction of higher cell alignment corresponding to the nematic director. The molecular organization inside the polymer determines such effects since no cell organization is observed using homeotropic or isotropic samples. These findings represent the first example of cellular alignment induced by the interaction with a nematic polymeric scaffold, setting the stage for new applications of liquid crystal polymers as active matter to control tissue growth.

Highly Tunable and Scalable Fabrication of 3D Flexible Graphene Micropatterns for Directing Cell Alignment.

Patterning graphene allows to precisely tune its properties to manufacture flexible functional materials or miniaturized devices for electronic and biomedical applications. However, conventional lithographic techniques are cumbersome for scalable production of time- and cost-effective graphene patterns, thus greatly impeding their practical applications. Here, we present a simple scalable fabrication of wafer-scale three-dimensional (3D) graphene micropatterns by direct laser tuning graphene oxide reduction and expansion using a LightScribe DVD writer. This one-step laser-scribing process can produce custom-made 3D graphene patterns on the surface of a disk with dimensions ranging from microscale up to decimeter scale in about 20 min. Through control over laser-scribing parameters, the resulting various 3D graphene patterns are exploited as scaffolds for controlling cell alignment. The 3D graphene patterns demonstrate their potential to biomedical applications, beyond the fields of electronics and photonics, which will allow to incorporate flexible graphene patterns for 3D cell or tissue culture to promote tissue engineering and drug testing applications.

A nano-fibrous platform of copolymer patterned surfaces for controlled cell alignment.

The last decade has witnessed great progress in understanding and manipulating self-assembly of block copolymers in solution. A wide variety of micellar structures can be created and many promising applications in bioscience have been reported. In particular, nano-fibrous micelles provide a great platform to mimic the filamentous structure of native extracellular matrix (ECM). However, the evaluation of this kind of filomicellar system with potential use in tissue engineering is virtually unexplored. The question behind it, such as if the block copolymer nano-fibrous micelles can regulate cellular response, has lingered for many years because of the difficulties in preparation and 3D manipulation of these tiny objects. Here, by using a combination approach of self-assembly of block copolymers and soft lithography, we establish a novel and unique nano-fibrous 2D platform of organized micelles and demonstrate that patterned micelles enable control over the cellular alignment behavior. The area density and orientation of fibrous micelles determine the alignment degree and directionality of cells, respectively. Furthermore, when cells were cultured on multi-directionally aligned micelles, a competitive response was observed. Due to the virtually infinite possibilities of functionalization of the micelle corona, our work opens a new route to further mimic the native fibrous networks with artificial micelles containing various functionalities.

Automated fabrication of hydrogel microfibers with tunable diameters for controlled cell alignment

A newly designed spinning device was utilized to produce continuous hydrogel microfibers with tunable diameters. It was found that the diameter of the microfiber was dependent on perfusion speed and coagulation wheel rotation rate. Their correlation was finally described by a mathematical expression, which proved to be useful for a size-tunable spinning technique. Based on the controllable fabrication of hydrogel microfibers with desired size, microfiber/cell-size-dependent cellular orientated spreading was studied by using PC12 and L929 as model cells. By further demonstrating the assembly of fibrous tissue-like grafts using the spun hydrogel microfibers, the wet spinning protocol was proved to be instructive for manufacturing size-tunable hydrogel microfibers, as well as two-dimensional or three-dimensional scaffolds with varied micro-structure for tissue engineering.

The control of cell orientation using biodegradable alginate fibers fabricated by near-field electrospinning

For spatially controlling cell alignment, near field electrospinning (NFES) was developed to direct-write alginate fiber patterns. Compared to randomly electrospun fibers, NFES fibers guided the extension of HEK 293T cells and the levels of cell alignment increased with decreasing fiber distances. However, these guiding fibers were unfavorable for cell adhesion and limited cell growth. To preserve cell alignment ability and improve biocompatibility, the stability of patterned alginate fibers was adjusted by regulating the level of ion crosslinking. These partially crosslinked NFES fibers demonstrated parallel line-patterns in the initial stage while gradually degraded with time. The reduction of fiber density increased the available area for cell growth and enhanced cell viability. On the other hand, aligned cells were still found on these degraded patterns, suggesting that cell morphologies were mainly guided during cell seeding. This dynamically controlled fiber pattern system fulfilled the need of controlling cell orientation and biocompatibility, thus was potential to modify scaffold surfaces for tissue engineering application.

Precisely parameterized experimental and computational models of tissue organization.

Patterns of cellular organization in diverse tissues frequently display a complex geometry and topology tightly related to the tissue function. Progressive disorganization of tissue morphology can lead to pathologic remodeling, necessitating the development of experimental and theoretical methods of analysis of the tolerance of normal tissue function to structural alterations. A systematic way to investigate the relationship of diverse cell organization to tissue function is to engineer two-dimensional cell monolayers replicating key aspects of the in vivo tissue architecture. However, it is still not clear how this can be accomplished on a tissue level scale in a parameterized fashion, allowing for a mathematically precise definition of the model tissue organization and properties down to a cellular scale with a parameter dependent gradual change in model tissue organization. Here, we describe and use a method of designing precisely parameterized, geometrically complex patterns that are then used to control cell alignment and communication of model tissues. We demonstrate direct application of this method to guiding the growth of cardiac cell cultures and developing mathematical models of cell function that correspond to the underlying experimental patterns. Several anisotropic patterned cultures spanning a broad range of multicellular organization, mimicking the cardiac tissue organization of different regions of the heart, were found to be similar to each other and to isotropic cell monolayers in terms of local cell-cell interactions, reflected in similar confluency, morphology and connexin-43 expression. However, in agreement with the model predictions, different anisotropic patterns of cell organization, paralleling in vivo alterations of cardiac tissue morphology, resulted in variable and novel functional responses with important implications for the initiation and maintenance of cardiac arrhythmias. We conclude that variations of tissue geometry and topology can dramatically affect cardiac tissue function even if the constituent cells are themselves similar, and that the proposed method can provide a general strategy to experimentally and computationally investigate when such variation can lead to impaired tissue function.

Tubular Scaffold with Shape Recovery Effect for Cell Guide Applications.

Tubular scaffolds with aligned polylactic acid (PLA) fibres were fabricated for cell guide applications by immersing rolled PLA fibre mats into a polyvinyl acetate (PVAc) solution to bind the mats. The PVAc solution was also mixed with up to 30 wt % β-tricalcium phosphate (β-TCP) content. Cross-sectional images of the scaffold materials obtained via scanning electron microscopy (SEM) revealed the aligned fibre morphology along with a significant number of voids in between the bundles of fibres. The addition of β-TCP into the scaffolds played an important role in increasing the void content from 17.1% to 25.3% for the 30 wt % β-TCP loading, which was measured via micro-CT (µCT) analysis. Furthermore, µCT analyses revealed the distribution of aggregated β-TCP particles in between the various PLA fibre layers of the scaffold. The compressive modulus properties of the scaffolds increased from 66 MPa to 83 MPa and the compressive strength properties decreased from 67 MPa to 41 MPa for the 30 wt % β-TCP content scaffold. The scaffolds produced were observed to change into a soft and flexible form which demonstrated shape recovery properties after immersion in phosphate buffered saline (PBS) media at 37 °C for 24 h. The cytocompatibility studies (using MG-63 human osteosarcoma cell line) revealed preferential cell proliferation along the longitudinal direction of the fibres as compared to the control tissue culture plastic. The manufacturing process highlighted above reveals a simple process for inducing controlled cell alignment and varying porosity features within tubular scaffolds for potential tissue engineering applications.