Biomaterial–Related Cell Microenvironment in Tissue Engineering and Regenerative Medicine

An appropriate cell microenvironment is key to tissue engineering and regenerative medicine.Revealing the factors that influence the cell microenvironment is a fundamental research topic in the fields of cell biology,biomaterials,tissue engineering,and regenerative medicine.The cell microenvironment consists of not only its surrounding cells and soluble factors,but also its extracellular matrix(ECM)or nearby external biomaterials in tissue engineering and regeneration.This review focuses on six aspects of bioma-terial-related cell microenvironments:①chemical composition of materials,②material dimensions and architecture,③material-controlled cell geometry,④effects of material charges on cells,⑤matrix stiff-ness and biomechanical microenvironment,and⑥surface modification of materials.The present chal-lenges in tissue engineering are also mentioned,and eight perspectives are predicted.

Study of Cell Behaviors on Anodized TiO_2 Nanotube Arrays with Coexisting Multi-Size Diameters

It has been revealed that the different morphologies of anodized TiO_2 nanotubes, especially nanotube diameters, triggered different cell behaviors. However, the influence of TiO_2 nanotubes with coexisting multi-size diameters on cell behaviors is seldom reported. In this work, coexisting four-diameter TiO_2 nanotube samples, namely,one single substrate with the integration of four different nanotube diameters(60, 150, 250, and 350 nm), were prepared by repeated anodization. The boundaries between two different diameter regions show well-organized structure without obvious difference in height. The adhesion behaviors of MC3T3-E1 cells on the coexisting fourdiameter TiO_2 nanotube arrays were investigated. The results exhibit a significant difference of cell density between smaller diameters(60 and 150 nm) and larger diameters(250 and 350 nm) within 24 h incubation with the coexistence of different diameters, which is totally different from that on the single-diameter TiO_2 nanotube arrays. The coexistence of four different diameters does not change greatly the cell morphologies compared with the singlediameter nanotubes. The findings in this work are expected to offer further understanding of the interaction between cells and materials.

Click and bioorthogonal hyaluronic acid hydrogels as an ultra-tunable platform for the investigation of cell-material interactions

The cellular microenvironment plays a major role in the biological functions of cells.Thus,biomaterials,especially hydrogels,which can be design to mimic the cellular microenvironment,are being increasingly used for cell encapsulation,delivery,and 3D culture,with the hope of controlling cell functions.Yet,much remains to be understood about the effects of cell-material interactions,and advanced synthetic strategies need to be developed to independently control the mechanical and biochemical properties of hydrogels.To address this challenge,we designed a new hyaluronic acid(HA)-based hydrogel platform using a click and bioorthogonal strain-promoted azide-alkyne cycloaddition(SPAAC)reaction.This approach facilitates the synthesis of hydrogels that are easy to synthesize and sterilize,have minimal swelling,are stable long term,and are cytocompatible.It provides bioorthogonal HA gels over an uncommonly large range of stiffness(0.5-45 kPa),all forming within 1-15 min.More importantly,our approach offers a versatile one-pot procedure to independently tune the hydrogel composition(e.g.,polymer and adhesive peptides).Using this platform,we investigate the independent effects of polymer type,stiffness,and adhesion on the secretory properties of human adipose-derived stromal cells(hASCs)and demonstrate that HA can enhance the secretion of immunomodulatory factors by hASCs.

Dynamic photoelectrical regulation of ECM protein and cellular behaviors

Dynamic regulation of cell-extracellular matrix(ECM)-material interactions is crucial for various biomedical applications.In this study,a light-activated molecular switch for the modulation of cell attachment/detachment behaviors was established on monolayer graphene(Gr)/n-type Silicon substrates(Gr/Si).Initiated by light illumination at the Gr/Si interface,pre-adsorbed proteins(bovine serum albumin,ECM proteins collagen-1,and fibronectin)underwent protonation to achieve negative charge transfer to Gr films(n-doping)throughπ-πinteractions.This n-doping process stimulated the conformational switches of ECM proteins.The structural alterations in these ECM interactors significantly reduced the specificity of the cell surface receptor-ligand interaction(e.g.,integrin recognition),leading to dynamic regulation of cell adhesion and eventual cell detachment.RNA-sequencing results revealed that the detached bone marrow mesenchymal stromal cell sheets from the Gr/Si system manifested regulated immunoregulatory properties and enhanced osteogenic differentiation,implying their potential application in bone tissue regeneration.This work not only provides a fast and feasible method for controllable cells/cell sheets harvesting but also gives new insights into the understanding of cell-ECM-material communications.

Micro and Nanofabrication methods to control cell-substrate interactions and cell behavior: A review from the tissue engineering perspective

Cell-substrate interactions play a crucial role in the design of better biomaterials and integration ofimplants with the tissues. Adhesion is the binding process of the cells to the substrate through interactionsbetween the surface molecules of the cell membrane and the substrate. There are severalfactors that affect cell adhesion including substrate surface chemistry, topography, and stiffness. Thesefactors physically and chemically guide and influence the adhesion strength, spreading, shape and fate ofthe cell. Recently, technological advances enabled us to precisely engineer the geometry and chemistry ofsubstrate surfaces enabling the control of the interaction cells with the substrate. Some of the mostcommonly used surface engineering methods for eliciting the desired cellular responses on biomaterialsare photolithography, electron beam lithography, microcontact printing, and microfluidics. Thesemethods allow production of nano- and micron level substrate features that can control cell adhesion,migration, differentiation, shape of the cells and the nuclei as well as measurement of the forces involvedin such activities. This review aims to summarize the current techniques and associate these techniqueswith cellular responses in order to emphasize the effect of chemistry, dimensions, density and design ofsurface patterns on cell-substrate interactions. We conclude with future projections in the field of cellsubstrateinteractions in the hope of providing an outlook for the future studies.

The influence of nanotopography on cell behaviour through interactions with the extracellular matrix-A review

Nanotopography presents an effective physical approach for biomaterial cell manipulation mediated through material-extracellular matrix interactions.The extracellular matrix that exists in the cellular microenvironment is crucial for guiding cell behaviours,such as determination of integrin ligation and interaction with growth factors.These interactions with the extracellular matrix regulate downstream mechanotransductive pathways,such as rearrangements in the cytoskeleton and activation of signal cascades.Protein adsorption onto nanotopography strongly influences the conformation and distribution density of extracellular matrix and,therefore,subsequent cell responses.In this review,we first discuss the interactive mechanisms of protein physical adsorption on nanotopography.Secondly,we summarise advances in creating nanotopographical features to instruct desired cell behaviours.Lastly,we focus on the cellular mechanotransductive pathways initiated by nanotopography.This review provides an overview of the current state-of-the-art designs of nanotopography aiming to provide better biomedical materials for the future.

Left-Right Symmetry or Asymmetry of Cells on Stripe-Like Micropatterned Material Surfaces

Material surfaces can induce cell responses such as contact guidance, yet little attention has been paid to further cell orientation. Herein, we report an interesting phenomenon of cell orientation beyond the classic contact guidance on a stripe-like micropattern with cell-adhesive arginineglycine-aspartate （RGD） peptides on a nonfouling background decorated by poly（ethylene glycol） （PEG）. Such a micropattern with cell adhesion contrast led to significant contact guidance after cell seeding. What is more, the localized and elongated cells were found to be further orientated out of the adhesive stripes, and even an anticlockwise rotation was observed for rat mesenchymal stem cells （rMSCs）. The left-right asymmetry of rMSCs stood only in statistics, for we observed all cases including clockwise orientation, anticlockwise orientation or just keeping the orientation of previous contact guidance. We further found that human foreskin fibroblasts （HFFs） preferred a clockwise rotation, while human mesenchymal stem cells （hMSCs） and human umbilical vascular endothelial cells （HUVECs） exhibited no significant preference to either direction, which indicated that the left-right symmetry or asymmetry was cell-type dependent. The present report has partially confirmed the cell chirality and revealed its complexity, calling for further careful and comprehensive investigation of the challenging topic of cell chirality on material surfaces.

3D additive-manufactured nanocomposite magnetic scaffolds: Effect of the application mode of a time-dependent magnetic field on hMSCs behavior

Over the past few years,the influence of static or dynamic magnetic fields on biological systems has become a topic of considerable interest.Magnetism has recently been implicated to play significant roles in the regulation of cell responses and,for this reason,it is revolutionizing many aspects of healthcare,also suggesting new opportunities in tissue engineering.The aim of the present study was to analyze the effect of the application mode of a time-dependent magnetic field on the behavior of human mesenchymal stem cells(hMSCs)seeded on 3D additivemanufactured poly(3-caprolactone)/iron-doped hydroxyapatite(PCL/FeHA)nanocomposite scaffolds.

Design and aligner-assisted fast fabrication of a microfluidic platform for quasi-3D cell studies on an elastic polymer

While most studies of mechanical stimulation of cells are focused on two-dimensional(2D)and three-dimensional(3D)systems,it is rare to study the effects of cyclic stretching on cells under a quasi-3D microenvironment as a linkage between 2D and 3D.Herein,we report a new method to prepare an elastic membrane with topographic microstructures and integrate the membrane into a microfluidic chip.The fabrication difficulty lay not only in the preparation of microstructures but also in the alignment and bonding of the patterned membrane to other layers.To resolve the problem,we designed and assembled a fast aligner that is cost-effective and convenient to operate.To enable quasi-3D microenvironment of cells,we fabricated polydimethylsiloxane(PDMS)microwell arrays(formed by micropillars of a few microns in diameter)with the microwell diameters close to the cell sizes.An appropriate plasma treatment was found to afford a coating-free approach to enable cell adhesion on PDMS.We examined three types of cells in 2D,quasi-3D,and 3D microenvironments;the cell adhesion results showed that quasi-3D cells behaved between 2D and 3D cells.We also constructed transgenic human mesenchymal stem cells(hMSCs);under cyclic stretching,the visualizable live hMSCs in microwells were found to orientate differently from in a 3D Matrigel matrix and migrate differently from on a 2D flat plate.This study not only provides valuable tools for microfabrication of a microfluidic device for cell studies,but also inspires further studies of the topological effects of biomaterials on cells.

Biomaterial nanotopography-mediated cell responses:experiment and modeling

The rapid development of fabrication and processing technologies in the past two decades has enabled researchers to introduce nanoscale features into materials which,interestingly,have been shown to greatly regulate the behavior and fate of biological cells.In particular,important cell responses(such as adhesion,proliferation,differentiation,migration,and filopodial growth)have all been correlated with material nanotopography.Given its great potential,intensive efforts have been made,both experimentally and theoretically,to understand why and how cells respond to nanoscale surface features,and this article reviews recent progress in this field.Specifically,a brief overview on the fabrication and modification techniques to create nanotopography on different materials is given first.After that,a summary of important experimental findings on the mediation of nanoscale surface topography on the behavior of various cells,as well as the underlying mechanism,is provided.Finally,both classical and recently developed approaches for modeling nanotopographymediated cell adhesion and filopodial growth are reviewed.