Role of nanotopography in the development of tissue engineered 3D organs and tissues using mesenchymal stem cells

Recent regenerative medicine and tissue engineering strategies(using cells, scaffolds, medical devices and gene therapy) have led to fascinating progress of translation of basic research towards clinical applications. In the past decade, great deal of research has focused on developing various three dimensional(3D) organs, such as bone, skin, liver, kidney and ear,using such strategies in order to replace or regenerate damaged organs for the purpose of maintaining or restoring organs' functions that may have been lost due to aging, accident or disease. The surface properties of a material or a device are key aspects in determining the success of the implant in biomedicine, as the majority of biological reactions in human body occur on surfaces or interfaces. Furthermore, it has been established in the literature that cell adhesion and proliferation are, to a great extent, influenced by the micro- and nanosurface characteristics of biomaterials and devices. In addition, it has been shown that the functions of stem cells, mesenchymal stem cells in particular, could be regulated through physical interaction with specific nanotopographical cues. Therefore, guided stem cell proliferation, differentiation and function are of great importance in the regeneration of 3D tissues and organs using tissue engineering strategies. This review will provide an update on the impact of nanotopography on mesenchymal stem cells for the purpose of developing laboratory-based 3D organs and tissues, as well as the most recent research and case studies on this topic.

Effects of nanotopography on stem cell phenotypes

Stem cells are unspecialized cells that can self renew indefinitely and differentiate into several somatic cells given the correct environmental cues.In the stem cell niche,stem cell-extracellular matrix(ECM)interactions are crucial for different cellular functions,such as adhesion,proliferation,and differentiation.Recently, in addition to chemical surface modifications,the importance of nanometric scale surface topography and roughness of biomaterials has increasingly becoming recognized as a crucial factor for cell survival and host tissue acceptance in synthetic ECMs.This review describes the influence of nanotopography on stem cell phenotypes.

Biophysical Regulation of Cell Behavior-Cross Talk between Substrate Stiffness and Nanotopography

The stiffness and nanotopographical characteristics of the extracellular matrix （ECM） influence numerous developmental, physiological, and pathological processes in vivo. These biophysical cues have therefore been applied to modulate almost all aspects of cell behavior, from cell adhesion and spreading to proliferation and differentiation. Delineation of the biophysical modulation of cell behavior is critical to the rational design of new biomaterials, implants, and medical devices. The effects of stiffness and topographical cues on cell behavior have previously been reviewed, respectively; however, the interwoven effects of stiffness and nanotopographical cues on cell behavior have not been well described, despite similarities in phenotypic manifestations. Herein, we first review the effects of substrate stiffness and nanotopography on cell behavior, and then focus on intracellular transmission of the biophysical signals from integrins to nucleus. Attempts are made to connect extracellular regulation of cell behavior with the biophysical cues. We then discuss the challenges in dissecting the biophysical regulation of cell behavior and in translating the mechanistic understanding of these cues to tissue engineering and regenerative medicine.

Nanotopography Impact in Shallow Trench Isolation Chemical Mechanical Polishing-Dependence on Slurry Characteristics

The nanotopography of the surface of silicon wafers has become an important issue in ULSI device manufacturing since it affects the post-chemical mechanical polishing (post-CMP) uniformity of the thickness deviation of dielectric films. In this study, the nanotopography impact was investigated in terms of its dependence on the characteristics of ceriabased slurries, such as the abrasive size, the grain size of the polycrystalline abrasive and the surfactant added to the slurry. It was found that the magnitude of the post-CMP oxide thickness deviation due to nanotopography increased with the surfactant concentration in the case of smaller abrasives but was almost independent of the concentration in the case of larger abrasives. The grain size of the polycrystalline abrasive did not affect the nanotopography impact.

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.

Surface modification of titanium implants with micro–nanotopography and NIR photothermal property for treating bacterial infection and promoting osseointegration

A double acid corrosion and subsequent hydrothermal treatment were used to fabricate a micro–nano-structured Ti substrates(Ti–M–N).Afterward,the mesoporous polydopamine(MPDA)nanoparticles as photothermal agent were prepared and immobilized on the surface of Ti–M–N samples,in order to obtain Ti–M–NMPDA sample.Unique micro–nanostructure properties and the photothermal effect of the modified Ti implant caused physical stress on the bacteria and the bacterial membrane damage,and eventually led to bacteria death.More importantly,based on excellent bioactivity and cytocompatibility of mussel-inspired materials,MPDA promoted adhesion,proliferation and osteogenic differentiation of mesenchymal stem cells in vitro.Furthermore,animal experiments in vivo further confirmed that the modified Ti implants could enhance osseointegration.

The influence, of nanotopography on organelle organization and communication

Cellular differentiation can be affected by the extracellular environment, particularly extracellular substrates. The nanotopography of the substrate may be involved in the mechanisms of cellular differentiation in vivo. Organelles are major players in various cellular functions; however, the influence of nano- topography on organelles has not yet been elucidated. In the present study, a micropit-nanotube topography （MNT） was fabricated on the titanium surface, and organelle-specific fluorescent probes were used to detect the intracellular organelle organization of MG63 cells. Communication between organelles, identified by organelle-specific GTPase expression, was evaluated by quantitative polymerase chain reaction and western blotting. Transmission electron microscopy was performed to evaluate the organelle structure. There were no significant differences in organelle distribution or number between the MNT and flat surface. However, organelle-specific GTPases on the MNT were dramatically downregulated. In addition, obvious endoplasmic reticulum lumen dilation was observed on the MNT surface, and the unfolded protein response （UPR） was also initiated. Regarding the relationships among organelle trafficking, UPR, and osteogenic differentiation, our findings may provide important insights into the signal transduction induced by nanotopography.

Controlling Osteogenic Differentiation through Nanoporous Alumina

Nanotopographical features are found to have significant effects on bone behavior. In the present study, nanoporous aluminas with different pore sizes (20, 100 and 200 nm in diameter), were evaluated for their osteoinductive and drug eluting properties. W20-17 marrow stromal cells were seeded on nanoporous alumina with and without the addition of BMP-2. Although cell proliferation was not affected by pore size, osteogenic differentiation was 200 nm as compared to 20 and 100 nm pores induced higher alkaline phosphatase activity (ALP) and osteocalcin expression levels, thus indicating osteoblastic differentiation. Cell morphology revealed that cells cultured on 20 nm pores adopted a rounded shape, while larger pores (200 nm) elicited an elongated morphology. Furthermore, ALP expression levels were consistently higher on BMP-2 loaded nanoporous alumina surfaces compared to unloaded surfaces, indicating that not only is nanoporous alumina osteoinductive, but also has the potential to be used as a drug eluting bone-implant coating.

Inorganic virus-like nanoparticles for biomedical applications:a minireview

Nature has the ingenious capability to design spiky topological features at the macro-and nanoscales,which exhibits fascinating interface adhesive properties by means of multivalent interactions.Following a biomimetic approach,such as nanoscale virus particles are highly infectious toward host cells,a range of organic and inorganic spiky particles(virus-like nanostructures)have been precisely engineered for diverse biomedical applications.Generally,organic virus-like particles(VLPs)derived from viral capsids(often termed as virosomes)have been extensively studied and reviewed,but concomitant concerns regarding immunogenicity and risks of mutagenesis limit clinical potential of organic VLPs.In contrast,inorganic VLPs(viral-mimicking topography)possess fascinating physicochemical characteristics,such as excellent electrical,optical,magnetic,mechanical and catalytic properties,which make them particularly suitable for biomedical applications.Alternatively,there is no comprehensive review related to inorganic VLPs engineered with non-viral shell for biomedical applications.Hence,in this review,we present a brief overview on inorganic VLPs,followed by summarizing the construction and properties of virus-like nanostructures,as well as the mechanisms of nano-bio interface interactions initiated by spiky topography.Furthermore,we focus on the recent advances of VLPs for biomedical applications(including biosensing,antibacterial therapy and cancer treatment).Finally,the future outlook and emerging challenges will be presented.This review aims to provide future scope of the rational design of inorganic non-viral vectors,especially with respect to gene-based therapy platforms.

Tunable nano-engineered anisotropic surface for enhanced mechanotransduction and soft-tissue integration

Electrochemically engineered titania(TiO_(2))nanopores enable tailored cellular function;however,the cellular mechanosensing mechanisms dictating the cell response and soft tissue integration are yet to be elucidated.Here,we report the fabrication of anisotropic TiO_(2)nanopores with diameters of 46 and 66 nm on microrough titanium(Ti)via electrochemical anodization,towards short-and long-term guidance of human primary gingival fibroblasts(hGFs).Cells on tissue culture plates and bare Ti substrates were used as controls.Notably,we show that nanopores with a diameter of 66 nm induced more mature focal adhesions of vinculin and paxillin at the membrane,encouraged the development of actin fibers at focal adhesion sites,led to elongated cell and nuclear shape.These topographical-driven changes were attributed to the Ras-related C3 botulinum toxin substrate 1(Rac 1)GTPase pathway and nuclear localisation of LAMIN A/C and yes-associated protein(YAP)and associated with increased ligament differentiation with elevated expression of the ligament marker Mohawk homeobox(MKX).Study findings reveal that minor tuning of nanopore diameter is a powerful tool to explore intracellular and nuclear mechanotransduction and gain insight into the relationships between nanomaterials and mechanoresponsive cellular elements.

Regulation of RAW 264.7 macrophages behavior on anodic TiO2 nanotubular arrays

Titanium （Ti） implants with TiO2 nanotubular arrays on the surface could regulate cells adhesion, proliferation and differentiation to determine the bone integra- tion. Additionally, the regulation of immune cells could improve osteogenesis or lead in appropriate immune reaction. Thus, we evaluate the behavior of RAW 264.7 macrophages on TiO2 nanotubular arrays with a wide range diameter （from 20 to 120 nm） fabricated by an electrochemical anodization process. In this work, the proliferation, cell viability and cytokine/chemokine secretion were evaluated by CCK-8, live/dead staining and ELISA, respectively. SEM and confocal microscopy were used to observe the adhesion morphology. Results showed that the small size nanotube surface was benefit for the macrophages adhesion and proliferation, while larger size surface could reduce the inflammatory response. These findings contribute to the design of immune-regulating Ti implants surface that supports successful implantation.

Enhanced bone regenerative properties of calcium phosphate ceramic granules in rabbit posterolateral spinal fusion through a reduction of grain size

Osteoinductivity is a crucial factor to determine the success and efficiency of posterolateral spinal fusion(PLF)by employing calcium phosphate(Ca-P)bioceramics.In this study,three kinds of Ca-P ceramics with microscale to nanoscale gain size(BCP-control,BCP-micro and BCP-nano)were prepared and their physicochemical properties were characterized.BCP-nano had the spherical shape and nanoscale gain size,BCP-micro had the spherical shape and microscale gain size,and BCP-control(BAM®)had the irregular shape and microscale gain size.The obtained BCP-nano with specific nanotopography could well regulate in vitro protein adsorption and osteogenic differentiation of MC3T3 cells.In vivo rabbit PLF procedures further confirmed that nanotopography of BCP-nano might be responsible for the stronger bone regenerative ability comparing with BCP-micro and BCP-control.Collectedly,due to nanocrystal similarity with natural bone apatite,BCP-nano has excellent efficacy in guiding bone regeneration of PLF,and holds great potentials to become an alternative to standard bone grafts for future clinical applications.

Cellular responses to nanoscale substrate topography of TiO_(2) nanotube arrays: cell morphology and adhesion

Nanotopographical features can be beneficial in augmenting cell functions and increasing osteogenic potential.However,the relationships between surface topographies and biological responses are difficult to establish due to the difficulty in controlling the surface topographical features at a low-nanometre scale.Herein,we report the fabrication of well-defined controllable titanium dioxide(TiO_(2))nanotube arrays with a wide range of pore sizes,30-175 nm in diameter,and use of the electrochemical anodization method to assess the effect of surface nanotopographies on cell morphology and adhesion.The results show that TiO_(2) nanotube arrays with pore sizes of 30 and 80 nm allowed for cell spreading of bone marrow-derived mesenchymal stem cells with increased cell area coverage.Additionally,cell adhesion was significantly enhanced by controlled nanotopographies of TiO_(2) nanotube arrays with 80 nm pore size.Our results demonstrate that surface modification at the nano-scale level with size tunability under controlled chemical/physical properties and culture conditions can greatly impact cell responses.These findings point to a new direction of material design for bone-tissue engineering in orthopaedic applications.

Regulation of osteoblast functions on titanium surfaces with different micro/nanotopographies and compositions

Surface modification of medical implants is considered as an effective method to improve cellular behaviors and the integration of tissues with materials. Titanium(Ti)-based materials with four different micro/nano-structures and compositions were prepared by acid etching, electrochemical anodization and alkali-heat treatment. The surface morphologies and compositions of the different surface-modified Ti materials were characterized by field-emission scanning electron microscopy(FE-SEM),atomic force microscopy(AFM) and X-ray diffraction(XRD). The effects of the micro/nano structured and compositions of the surfaces on cellular responses were investigated in vitro by observing the morphology, adhesion, proliferation and osteogenic differentiation of osteoblasts. To further investigate the underlying mechanisms, an RT-PCR assay was performed to analyze the expression levels of cell adhesion-related genes. Our results indicated that the nanosized structure and anatase composition could promote the adhesion and proliferation of MC3T3-E1 pre-osteoblast, as well as alkaline phosphatase activity and extracellular matrix mineralization via the integrin-FAK signaling pathway. Taken together, our innovation presented in this work demonstrated that the surface nano-structure design and composition of biomedical implants can be modified of for future orthopaedic applications.