Disordered Graphene/Quartz Fabric as Biocompatible and Conductive Scaffold Promising for Regulated Growth and Differentiation of Nerve Cells

Nowadays, the use of topographical features and electrical conductivity of scaffolds at the cell-substrate interface for effectively regulating cell growth and differentiation have gained increasing attention due to great demands for tissue engineering. Herein, a facile approach to the growth of highly disordered graphene nanosheets (HDGNs) is demonstrated on a cheap and weaving quartz-braided structure as a functionalized scaffold for the differentiation of nerve cells. The patterned aligned structure can effectively integrate the advantages of a conductive graphene-functional interface (favorable for cell attachment and growth), topologically woven surface structure, providing a flexible and multifunctional regulatory platform for nerve cell growth. Compared with monocrystal polycrystalline graphene, amorphous graphene has high biocompatibility due to sufficient active sites, and has high conductivity to the composite nonconductive substrate, which can realize electrical stimulation (ES) of cell differentiation. Herein, the HDGN/quartz fabric with high biocompatibility (the cell viability is 98%), and great electrical conductivity, is proved. Then, the applied ES coupled with HDGN/quartz fabric significantly enhances selective neuronal differentiation into neurons (the differentiation growth rate is 131%). Collectively, herein, a new material basis is provided for electric induction of cell growth and differentiation, providing more possibilities for the development of intelligent biological applications.     

Synergistic control of mesenchymal stem cell differentiation by nanoscale surface geometry and immobilized growth factors on TiO2 nanotubes

The aim of this study is to elucidate whether combined environmental signals provided by nanoscale topography and by growth factors control cell behavior of mesenchymal stem cells (MSCs) in a synergistic or simply additive manner. Chondrogenic and osteogenic differentiation of MSCs is studied on vertically aligned TiO(2) nanotubes of size 15 and 100 nm with and without immobilized bone morphogenetic protein-2 (BMP-2). Although BMP-2 coating stimulates both chondrogenic and osteogenic differentiation of MSCs, the response strongly depends on the surface nanoscale geometry of the BMP-2-coated nanotubes. Chondrogenic differentiation is strongly supported on 100 nm BMP-2-coated nanotubes, but not on 15 nm nanotubes, which induce spreading and de-differentiation of chondrocytes. A similar response is observed with primary chondrocytes, which maintain their chondrogenic phenotype on BMP-2-coated 100 nm nanotubes, but de-differentiate on 15 nm nanotubes. In contrast, osteogenic differentiation is greatly enhanced on 15 nm but not on 100 nm BMP-2-coated nanotubes as shown previously. Furthermore, covalent immobilization of BMP-2 rescues MSCs from apoptosis occurring on uncoated 100 nm TiO(2) nanotube surfaces. Thus, combined signals provided by BMP-2 immobilized to a defined lateral nanoscale spacing geometry seem to contain environmental cues that are able to modulate a lineage-specific decision of MSC differentiation and cell survival in a synergistic manner.     

Patterning the mechanical properties of hydrogen silsesquioxane films using electron beam irradiation for application in mechano cell guidance

Hydrogen silsesquioxane (HSQ) is a material with the potential for studying the effect of surface stiffness on stem cell differentiation. Here, the effects of electron beam dose on the topography and the mechanical properties of HSQ obtained with or without trimethylamine (TMA) development are characterised by atomic force microscopy imaging and indentation. A correlation between the surface stiffness (uniform across the sample) and electron beam exposure is observed. Surface roughness of HSQ samples developed in TMA decreases exponentially with increasing electron beam exposure. Surface coating with plasma polymerised allylamine (ppAAm) leads to an overall decrease in stiffness values. However, the increase in surface stiffness with increasing electron beam exposure is still evident. The ppAAm coating is shown to facilitate human mesenchymal stem cell adhesion.     

Differentiation of human mesenchymal stem cells on plasma-treated polyetheretherketone

Polyetheretherketone (PEEK) generally exhibits physical and chemical characteristics that prevent osseointegration. To activate the PEEK surface, we applied oxygen and ammonia plasma treatments. These treatments resulted in surface modifications, leading to changes in nanostructure, contact angle, electrochemical properties and protein adhesion in a plasma power and process gas dependent way. To evaluate the effect of the plasma-induced PEEK modifications on stem cell adhesion and differentiation, adipose tissue-derived mesenchymal stem cells (adMSC) were seeded on PEEK specimens. We demonstrated an increased adhesion, proliferation, and osteogenic differentiation of adMSC in contact to plasma-treated PEEK. In dependency on the process gas (oxygen or ammonia) and plasma power (between 10 and 200 W for 5 min), varying degrees of osteogenic differentiation were induced. When adMSC were grown on 10 and 50 W oxygen and ammonia plasma-treated PEEK substrates they exhibited a doubled mineralization degree relative to the original PEEK. Thus plasma treatment of PEEK specimens induced changes in surface chemistry and topography and supported osteogenic differentiation of adMSC in vitro. Therefore plasma treated PEEK holds perspective for contributing to osseointegration of dental and orthopedic load-bearing PEEK implants in vivo.     

Differentiation of human mesenchymal stem cells on nano- and micro-grain size titania

The current study investigated the osteogenic differentiation of human mesenchymal stem cells cultured on titania surfaces with grain sizes ranging from 50 to 1500 nm in either control or osteogenic medium. Characterization of osteogenic differentiation included quantification of the osteopontin and alkaline phosphatase expression by the cells, as well as of the content of calcium in the extracellular matrix. Mesenchymal stem cell differentiation was not observed on any of the grain sizes tested without dexamethasone and osteogenic-stimulating chemical agents (specifically, ascorbic acid and beta-glycerolphosphate) in the culture medium. Little-to-no mesenchymal stem cell differentiation was detected on the 50 nm substrates under osteogenic media. In contrast, osteogenic differentiation occurred earlier, and to greater extent, on the 200 nm grain size titania, compared to results obtained on either the 50 or 1500 nm grain sizes, or the glass (reference) surfaces, under osteogenic media. These results demonstrated that biomaterial substrate topography, such as ceramic grain size, affects mesenchymal stem cell differentiation in a size-dependent but, non-linear, manner.     

Hierarchically engineered fibrous scaffolds for bone regeneration

Surface properties of biomaterials play a major role in the governing of cell functionalities. It is well known that mechanical, chemical and nanotopographic cues, for example, influence cell proliferation and differentiation. Here, we present a novel coating protocol to produce hierarchically engineered fibrous scaffolds with tailorable surface characteristics, which mimic bone extracellular matrix. Based on the sol–gel method and a succession of surface treatments, hollow electrospun polylactic acid fibres were coated with a silicon–calcium–phosphate bioactive organic–inorganic glass. Compared with pure polymeric fibres that showed a completely smooth surface, the coated fibres exhibited a nanostructured topography and greater roughness. They also showed improved hydrophilic properties and a Young''s modulus sixfold higher than non-coated ones, while remaining fully flexible and easy to handle. Rat mesenchymal stem cells cultured on these fibres showed great cellular spreading and interactions with the material. This protocol can be transferred to other structures and glasses, allowing the fabrication of various materials with well-defined features. This novel approach represents therefore a valuable improvement in the production of artificial matrices able to direct stem cell fate through physical and chemical interactions.     

Development of human mesenchymal stem cells on DC sputtered titanium nitride thin films

The biocompatible properties of titanium nitride (TiN) have opened a new field of applications for this material. In the present work, TiN coatings with thicknesses around 1 m have been prepared by DC magnetron sputtering. The aim has been to evaluate the adherence, growth and proliferation of human pluripotent mesenchymal stem cells (hMSCs) on the surface of TiN films with contrasted structural, electrical, and mechanical properties. For this purpose, the films were characterized by X-ray diffraction, scanning electron microscopy, sheet resistance measurements, and nanoindentation. Biological tests show that hMSCs adhere and proliferate onto TiN surfaces. The combination of the mechanical, electrical, and biological responses suggest that TiN coatings present appropriate properties to induce the in vitro stimulated differentiation of hMSCs. This possibility gives an added value to TiN based biomaterial coatings.     

超顺排碳纳米管/硅橡胶复合材料各向异性的电学性能和力学性能

采用直接浸润法制备了具有不同层数的超顺排碳纳米管(SACNT)薄膜与硅橡胶的复合材料,使碳纳米管薄膜能够在硅橡胶基体表面均匀分散。测量了SACNT薄膜/硅橡胶复合材料在各个方向的导电性能和力学性能,研究了影响复合材料导电性和力学性能的因素。实验结果表明:SACNT薄膜/硅橡胶复合材料的导电性和杨氏模量都随着碳纳米管薄膜厚度的增加而增加,且具有显著的各向异性。垂直于碳纳米管排列方向的电阻率平均比平行方向的大一个数量级。当碳纳米管层数为240层时,平行于碳纳米管排列方向的杨氏模量为116.9 MPa(比纯硅橡胶基体增加了142倍),而垂直方向的杨氏模量仅为1.23 MPa(比纯硅橡胶基体增加50%),两者之间相差近100倍。结果表明,可以通过选择不同的参数,获得具有特定导电性和杨氏模量的SACNT薄膜/硅橡胶复合材料,并在实际中加以应用。     

Evaluation of early stage human bone marrow stromal proliferation, cell migration and osteogenic differentiation on μ-MIM structured stainless steel surfaces

It is well established that surface topography greatly affect cell—surface interactions. In a recent study we showed that microstructured stainless steel surfaces characterized by the presence of defined hexagonally arranged hemisphere-like structures significantly affected cell architecture (shape and focal adhesion size) of primary human bone mesenchymal stromal cells. This study aimed at further investigating the influence these microstructures (microcline protruding hemispheres) on critical aspects of cell behaviour namely; proliferation, migration and osteogenic differentiation. As with previously reported data, we used primary human bone mesenchymal stromal cells to investigate such effects at an early stage in vitro. Cells of different patients were utilised for cell migration studies. Our data showed that an increase in cell proliferation was exhibited as a function of surface topography (hemispheres). Cell migration velocity also varied as a function of surface topography on patient specific basis and seems to relate to the differentiated state of the seeded cell population (as demonstrated by bALP positivity). Osteogenic differentiation, however, did not exhibit significant variations (both up and down-regulation) as a function of both surface topography and time in culture.     

Surface modification of polydimethylsiloxane (PDMS) induced proliferation and neural-like cells differentiation of umbilical cord blood-derived mesenchymal stem cells

Stem cell-based therapy has recently emerged for use in novel therapeutics for incurable diseases. For successful recovery from neurologic diseases, the most pivotal factor is differentiation and directed neuronal cell growth. In this study, we fabricated three different widths of a micro-pattern on polydimethylsiloxane (PDMS; 1, 2, and 4 microm). Surface modification of the PDMS was investigated for its capacity to manage proliferation and differentiation of neural-like cells from umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). Among the micro-patterned PDMS fabrications, the 1 microm-patterned PDMS significantly increased cell proliferation and most of the cells differentiated into neuronal cells. In addition, the 1 microm-patterned PDMS induced an increase in cytosolic calcium, while the differentiated cells on the flat and 4 microm-patterned PDMS had no response. PDMS with a 1 microm pattern was also aligned to direct orientation within 10 degrees angles. Taken together, micro-patterned PDMS supported UCB-MSC proliferation and induced neural like-cell differentiation. Our data suggest that micro-patterned PDMS might be a guiding method for stem cell therapy that would improve its therapeutic action in neurological diseases.     

Fabrication of carboxylic graphene oxide-composited polypyrrole film for neurite growth under electrical stimulation

An aligned composite film was fabricated via the deposition of carboxylic graphene oxide (C-GO) and polypyrrole (PPy) nanoparticles on aligned poly(L-lactic acid) (PLLA) fiber-films (named as C-GO/PPy/PLLA), which has the core (PLLA)–sheath (C-GO/PPy) structure, and the composition of C-GO (~4.8 wt.% of PPy sheath) significantly enhanced the tensile strength and the conductivity of the PPy/PLLA film. Especially, after 4 weeks of immersion in the PBS solution, the conductivity and the tensile strength of C-GO/PPy/PLLA films still remained ~6.10 S/cm and 28.9 MPa, respectively, which could meet the need of the sustained electrical stimulation (ES) therapy for nerve repair. Moreover, the neurite length and the neurite alignment were significantly increased through exerting ES on C-GO/PPy/PLLA films due to their sustained conductivity in the fluid of cell culture. These results indicated that C-GO/PPy/PLLA with sustained conductivity and mechanical property possessed great potential of nerve repair by exerting lasting-ES.     

Tailoring RGD local surface density at the nanoscale toward adult stem cell chondrogenic commitment

Arginine-glycine-aspartic acid (RGD) dendrimer-based nanopatterns on poly(L-lactic acid) were used as bioactive substrates to evaluate the impact of the RGD local surface density on the chondrogenic induction of adult human mesenchymal stem cells.During chondrogenic commitment,active extracellular matrix (ECM) remodeling takes place,playing an instructive role in the differentiation process.Although three-dimensional environments such as pellet or micromass cultures are commonly used for in vitro chondrogenic differentiation,these cultures are rather limited with respect to their ability to interrogate cells in cell-ECM interactions.In the present study,the nanopatterns of the tunable RGD surface density were obtained as a function of the initial dendrimer concentration.The local RGD surface density was quantified through probability contour plots for the minimum interparticle distance,constructed from the corresponding atomic force microscopy images,and correlated with the cell adhesion and differentiation response.The results revealed that the local RGD surface density at the nanoscale acts as a regulator of chondrogenic commitment,and that intermediate adhesiveness of cells to the substrates favors mesenchymal cell condensation and early chondrogenic differentiation.     

Clay Nanotubes Aligned with Shear Forces for Mesenchymal Stem Cell Patterning

Aligned halloysite nanotubes on solid substrates are fabricated by a shearing method with brush assistance. These clay nanotubes are aligned by shear force in strip‐like patterns accomplished with drying ordering at elevated temperatures. The nanotubes' orientation is governed by “coffee‐ring” formation mechanisms depending on the dispersion concentration, nanotube charge, and speed of thermos‐evaporation. Polarized light irradiated through the patterns demonstrates birefringence and confirms the orientation. Scanning electron microscopy and atomic force microscopy show that the nanotubes are aligned along the direction of the wetting lines above 4 wt%, while they are not oriented at lower concentrations. Halloysite concentration, drying temperature, and type of brush fibers affect the pattern ordering. The aligned halloysite systems on glass, tissue culture plates, and polymer films, provide a promising platform for biocell guiding. Human foreskin fibroblasts proliferated well on the aligned clay patterns and the cell orientation agrees with the nanotube direction. Human bone mesenchymal stem cells (HBMSCs) are also cultured on the organized halloysite coating. The clay patterns support HBMSC proliferation with alignment, and such nanostructured substrates promote osteogenesis differentiation without growth factors. This facile method for preparing aligned halloysite patterns on solid substrates is very promising for surface modification in biotissue engineering.     

The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder

A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.     

The anatase phase of nanotopography titania plays an important role on osteoblast cell morphology and proliferation

The surface properties of biomaterials play a vital role in cell morphology and behaviors such as cell adhesion, migration, proliferation and differentiation. Three different crystal phases of titania film (rutile, anatase and amorphous titania) with similar roughness were successfully synthesized by DC reactive magnetron sputtering. The surface roughness of each film was about 8-10 nm. Primary rat osteoblasts were used to observe changes in morphology and to evaluate cell behavior at the film surface. The number of the osteoblasts on anatase film was significantly higher than rutile and amorphous films after 36 and 72 h incubation. More importantly, synthesis of alkaline phosphatase was significantly greater by osteoblasts cultured on anatase film than on rutile and amorphous films after 7 and 14 days. In addition, the cells grown on the anatase phase film had the largest spreading area; the actin filaments in cells with regular directions were well defined and fully spreaded. The results indicate that the anatase phase of titania with nanoscale topography yield the best biological effects for cell adhesion, spreading, proliferation and differentiation. There are strong therapeutic prospects for this biomaterial film for osteoblast proliferation, with possible applications for orthopedic and dental implant.     

The fabrication of biomineralized fiber-aligned PLGA scaffolds and their effect on enhancing osteogenic differentiation of UCMSC cells

The key factor of scaffold design for bone tissue engineering is to mimic the microenvironment of natural bone extracellular matrix (ECM) and guide cell osteogenic differentiation. The biomineralized fiber-aligned PLGA scaffolds (a-PLGA/CaPs) was developed in this study by mimicking the structure and composition of native bone ECM. The aligned PLGA fibers was prepared by wet spinning and then biomineralized via an alternate immersion method. Introduction of a bioceramic component CaP onto the PLGA fibers led to changes in surface roughness and hydrophilicity, which showed to modulate cell adhesion and cell morphology of umbilical cord mesenchymal stem cells (UCMSCs). It was found that organized actin filaments of UCMSCs cultured on both a-PLGA and a-PLGA/CaP scaffolds appeared to follow contact guidance along the aligned fibers, and those cells grown on a-PLGA/CaP scaffolds exhibited a more polarized cellular morphology. The a-PLGA/CaP scaffold with multicycles of mineralization facilitated the cell attachment on the fiber surfaces and then supported better cell adhesion and contact guidance, leading to enhancement in following proliferation and osteogenic differentiation of UCMSCs. Our results give some insights into the regulation of cell behaviors through design of ECM-mimicking structure and composition and provide an alternative wet-spun fiber-aligned scaffold with HA-mineralized layer for bone tissue engineering application.     

Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

Abstract

Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.     

ITO平面微电极上P(VDF-TrFE)薄膜的制备及性能表征

在ITO平面微电极上设计了P(VDF-TrFE)纳米级薄膜,以构建仅电场作用的模式,并避免其他因素的干扰.通过旋涂法制备了不同厚度的P(VDF-TrFE)薄膜,当厚度达到470 nm时,薄膜具有足够的致密度和绝缘性,能隔绝电极上的微电流和可能产生的电化学产物.由于电极上覆有薄膜,在生物电刺激的应用中,还可消除电极材料不...     

Adipose-derived stem cells could sense the nano-scale cues as myogenic-differentiating factors

Microenvironmental cues, such as surface topography and substrate stiffness, may affect stem cells adhesion, morphology, alignment, proliferation and differentiation. Adipose derived stem cells (ASCs) have attracted considerable interest in regenerative medicine due to their easy isolation, extensive in vitro expandability and ability to differentiate along a number of different tissue-specific lineages. The aim of this work was to investigate ASCs adhesion, alignment and differentiation into myogenic lineage on nanofibrous polymeric scaffolds with anisotropic topography. Nanostructured scaffolds with randomized or parallel fibers were fabricated by electrospinning using polycaprolactone (PCL) and the polycarbonate-urethane ChronoFlex AL 80A (CFAL). Cells expressed myosin (fast skeletal) and tropomyosin in all surface topographies 7 days after seeding but myotube formation was only observed on CFAL scaffolds and only few myotubes were formed on PCL scaffolds. The different cell behavior could be ascribed to two main parameters: fibers dimensions and fibers orientation of the substrates that could result in a better myotube formation on CFAL scaffolds.     

Fabrication and in vitro evaluation of stable collagen/hyaluronic acid biomimetic multilayer on titanium coatings

Layer-by-layer (LBL) self-assembly technique has been proved to be a highly effective method to immobilize the main components of the extracellular matrix such as collagen and hyaluronic acid on titanium-based implants and form a polyelectrolyte multilayer (PEM) film by electrostatic interaction. However, the formed PEM film is unstable in the physiological environment and affects the long-time effectiveness of PEM film. In this study, a modified LBL technology has been developed to fabricate a stable collagen/hyaluronic acid (Col/HA) PEM film on titanium coating (TC) by introducing covalent immobilization. Scanning electron microscopy, diffuse reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to characterize the PEM film. Results of Sirius red staining demonstrated that the chemical stability of PEM film was greatly improved by covalent cross-linking. Cell culture assays further illustrated that the functions of human mesenchymal stem cells, such as attachment, spreading, proliferation and differentiation, were obviously enhanced by the covalently immobilized Col/HA PEM on TCs compared with the absorbed Col/HA PEM. The improved stability and biological properties of the Col/HA PEM covalently immobilized TC may be beneficial to the early osseointegration of the implants.