Clinical potential of plasma-functionalized graphene oxide ultrathin sheets for bone and blood vessel regeneration: Insights from cellular and animal models.

Graphene and graphene oxide (GO), due to their unique chemical and physical properties, possess biochemical characteristics that can trigger intercellular signals promoting tissue regeneration. Clinical applications of thin GO-derived sheets have inspired the development of various tissue regeneration and repair approaches. In this study, we demonstrate that ultrathin sheets of plasma-functionalized and reduced GO, with the oxygen content ranging from 3.2 % to 22 % and the nitrogen content from 0 % to 8.3 %, retain their essential mechanical and molecular integrity, and exhibit robust potential for regenerating bone tissue and blood vessels across multiple cellular and animal models. Initially, we observed the growth of blood vessels and bone tissue in vitro using these functionalized GO sheets on human adipose-derived mesenchymal stem cells and umbilical vein endothelial cells. Remarkably, our study indicates a 2.5-fold increase in mineralization and two-fold increase in tubule formation even in media lacking osteogenic and angiogenic supplements. Subsequently, we observed the initiation, conduction, and formation of bone and blood vessels in a rat tibial osteotomy model, evident from a marked 4-fold increase in the volume of low radio-opacity bone tissue and a significant elevation in connectivity density, all without the use of stem cells or growth factors. Finally, we validated these findings in a mouse critical-size calvarial defect model (33 % higher healing rate) and a rat skin lesion model (up to 2.5-fold increase in the number of blood vessels, and 35 % increase in blood vessels diameter). This study elucidates the pro-osteogenic and pro-angiogenic properties of both pristine and plasma-treated GO ultrathin films. These properties suggest their significant potential for clinical applications, and as valuable biomaterials for investigating fundamental aspects of bone and blood vessel regeneration.

Graphene and graphene oxide for bio-sensing: General properties and the effects of graphene ripples.

The use of Graphene based materials, such as graphene oxide (GO), in biosensing applications is gaining significant interest, due to high signal output, with strong potential for high industrial growth rate. Graphene's excellent conduction and mechanical properties (such as toughness and elasticity) coupled with high reactivity to chemical molecules are some of its appealing properties. The presence of ripples on the surface (whether indigenous or induced) represents another property/variable that provide enormous potential if harnessed properly. In this article, we review the current knowledge regarding the use of graphene for biosensing. We discuss briefly the general topic of using graphene for biosensing applications with special emphasis on wearable graphene-based biosensors. The intrinsic ripples of graphene and their effect on graphene biosensing capabilities are thoroughly discussed. We dedicate a section also for the manipulation of intrinsic ripples. Then we review the use of Graphene oxide (GO) in biosensing and discuss the effect of ripples on its properties. We present a review of the current biosensor devices made out of GO for detection of different molecular targets. Finally, we present some thoughts for future perspectives and opportunities of this field. STATEMENT OF SIGNIFICANCE: Biosensors are tools that detect the presence and amount of a chemical substance, such as pregnancy tests and glucose monitoring devices. They are general portable, have short response times and are sensitive, making them highly effective. Gold and silver are used in biosensors and more recently, graphene.  Graphene is sheets of carbon atoms and is the only two-dimensional crystal in nature. It has unique features allowing its effective use in biosensing applications, including the presence of ripples (non-flat areas that give it its electronic properties). The last comprehensive review of this topic was published in 2016. This paper reviews the current knowledge of graphene based biosensors, with a focus on ripples and their effect on graphene biosensing capabilities.

Fibronectin/thermo-responsive polymer scaffold as a dynamic ex vivo niche for mesenchymal stem cells.

In this paper, we created a dynamic adhesive environment (DAE) for adipose tissue-derived mesenchymal stem cells (ADMSCs) cultured on smart thermo-responsive substrates, i.e., poly (N-isopropyl acrylamide) (PNIPAM), via introducing periodic changes in the culture temperature. We further explored the particular role of adsorbed fibronectin (FN), an important cell adhesive protein that was recently attributed to the recruitment of stem cells in the niche. The engineered FN/PNIPAM DAE system significantly increased the symmetric renewal of ADMSCs, particularly between passages 7 and 9 (p7-p9), before it dropped down to the level of the control (FN-coated TC polystyrene). This decline in the growth curve was consistent with the increased number of senescent cells, the augmented average cell size and the suppressed FN matrix secretion at late passages (p10-p12), all of them characteristic for stem cells ageing, which equivocally tended to slow down at our DAE system. FN supported also the osteogenic response of ADMSCs (apart from the previous observations with plain PNIPAM substrata) indicated by the significant increase of alkaline phosphatase (ALP) activity at days 7 and 14. The minimal changes in the Ca deposition, however, suggest a restricted effect of DAE on the early osteogenic response of ADMSCs only. Thus, the engineering of niche-like DAE involving FN uncovers a new tissue engineering strategy for gaining larger amounts of functionally active stem cells for clinical application.

Enhanced Osteogenic Differentiation of Human Primary Mesenchymal Stem and Progenitor Cultures on Graphene Oxide/Poly(methyl methacrylate) Composite Scaffolds.

Due to its versatility, small size, large surface area, and ability to interact with biological cells and tissues, graphene oxide (GO) is an excellent filler for various polymeric composites and is frequently used to expand their functionality. Even though the major advantage of the incorporation of GO is the enhancement of mechanical properties of the composite material, GO is also known to improve bioactivity during biomineralization and promote osteoblast adhesion. In this study, we described the fabrication of a composite bone cement made of GO and poly(methyl methacrylate) (PMMA), and we investigated its potential to enhance osteogenic differentiation of human primary mesenchymal stem and progenitor cells. Through the analysis of three differentiation markers, namely alkaline phosphatase, secreted protein acidic and rich in cysteine, and bone morphogenetic protein-2 in the presence and in the absence of an osteogenic differentiation medium, we were able to indicate a composite produced manually with a thick GO paper as the most effective among all investigated samples. This effect was related to its developed surface, possessing a significant number of voids and pores. In this way, GO/PMMA composites were shown as promising materials for the applications in bone tissue engineering.

Establishing multiple osteogenic differentiation pathways of mesenchymal stem cells through different scaffold configurations.

Mimicking the complex organization of the extracellular matrix (ECM), especially its structure and dimensionality, is necessary to produce living tissues from stem cells. In compliance with a previously established role of nanofiber organization for the osteogenic differentiation of stem cells, here we used hybrid fibrinogen/poly(l-lactide-ε-caprolactone) (FBG/PLCL) nanofibers arranged in aligned and honeycomb configurations, to recapitulate the highly oriented ECM of the cortical bone and the sponge-like (i.e., honeycomb) environment of the cancellous one, respectively. Using special bilayered constructs, we demonstrate that the dimensionality (i.e., 2D vs. 3D) of the nanofibers as well as their architecture (i.e., honeycomb vs. aligned) affects differently the overall morphology and the expression of multiple osteogenic genes of human adipose-derived mesenchymal stem cells (ADMSCs). The cells had elongated shape with markedly increased cell mobility when seeded on aligned nanofibers. Conversely, on honeycomb-shaped nanofibers, ADMSCs initially concentrated inside the honeycomb curvatures adopting rounded morphology, but late, they formed network-like structures overlaying the honeycomb curvatures. By employing quantitative polymerase chain reaction (qPCR), we further show that a 3D environment generally supports the multiple osteogenic response of ADMSCs, but honeycomb and aligned architectures promote rather different differentiation pathways.

Effect of nitrogen plasma treatment on the crystallinity and self-bonding of polyetheretherketone (PEEK) for biomedical applications.

Polyetheretherketone (PEEK) is a thermoplastic material with outstanding properties and high potential for biomedical applications, including hermetic encapsulation of active implantable devices. Different biomedical grade PEEK films with initial degree of crystallinity ranging from 8% to 32% (with or without mineral filling) were inspected. PEEK surfaces were treated with nitrogen RF plasma and the effects on materials crystallinity and self-bonding were evaluated. In particular, the relationship between auto-adhesive properties and crystalline content of PEEK before and after plasma treatment was examined. PEEK samples showed different bonding strength depending on their degree of crystallinity, with higher self-bonding performance of mineral-filled semi-crystalline films. XRD did not show any modification of the PEEK microstructure as a result of plasma treatment, excluding a significant influence of crystallinity on the self-bonding mechanisms. Nevertheless, plasma surface treatment successfully improved the self-bonding strength of all the PEEK films tested, with larger increase in the case of semi-crystalline unfilled materials. This could be interpreted to the increase in chain mobility that led to interfacial interpenetration of the amorphous phase.

Friction and Adhesion of Different Structural Defects of Graphene.

Graphene structural defects, namely edges, step-edges, and wrinkles, are susceptible to severe mechanical deformation and stresses under tribo-mechanical operations. Applied forces may cause deformation by folding, buckling, bending, and tearing of these defective sites of graphene, which lead to a remarkable decline in normal and friction load bearing capacity. In this work, we experimentally quantified the maximum sustainable normal and friction forces, corresponding to the damage thresholds of the different investigated defects as well as their pull-out (adhesion) forces. Horizontal wrinkles (with respect to the basal plane, i.e., folded) sustained the highest normal load, up to 317 nN, during sliding, whereas for vertical (i.e., standing) wrinkles, step-edges, and edges, the load bearing capacities are up to 113, 74, and 63 nN, respectively. The related deformation mechanisms were also experimentally investigated by varying the normal load up to the initiation of the damage from the defects and extended with the numerical results from molecular dynamics and finite element method simulations.

Surface modification and characterization of GO/polymer thin coatings as excellent bio-active platforms for tissue regeneration.

Osteo-integration and tissue regeneration are vital for the longevity, durability, and unremitting functionality of medical implants/scaffolds implanted in vivo. It's essential for biomaterials used for in vivo implantation to induce the cellular secretion of growth factors, necessary for the desired tissue generation, since the administration of artificial growth factors, in vivo, is largely prohibited. Plasma functionalized (N2 and O2) and stabilized Graphene Oxide (GO) thin layers in a hybrid with amorphous carbon (aC) induced the expression of vascular endothelial growth factor (VEGF) and osteoprotegerin (OPG) growth factors in fibroblasts (hGF) and, more remarkably, in osteoblasts (hFOB) cells confirming the suitability for tissue regeneration and osteo-integration applications. We also observed a negative trend between hGF fibroblasts, but not hFOB osteoblasts, cellular viability and GO presence in the hybrid films that might indicate the phenomenon of oxidative stress. We traced that back to the presence of higher concentrations of carboxyl and the carbonyl groups on the surface of the GO rich coatings. The above described properties provided by GO coatings might be desirable for bio-selectivity applications and for the reduction of the undesired fibrosis process that is associated with medical implants in vivo environment. Moreover, novel plasma functionalized GO/polymer hybrid thin coating hybrid compositions are promising candidates for tissue engineering and bioengineering applications as excellent antimicrobial and anticancer platforms.

Lab-on-a-chip device made by autohesion-bonded polymers.

Polymers have the obvious advantages of flexibility in design and cost effectiveness to fabricate a lab-on-a-chip (LOC) device. Polyether ether ketone (PEEK) in particular is very attractive choice as it adds biocompatibility in addition to the possibility of hematic sealing in a 3D design. Hereby, we extend our previous successful technology of autohesive hermetic bonding of medical implants into lab-on-a-chip devices. We explore a conceptual 3D micro channels design with hermetic potential using PEEK and PS sheets. A hermetic and mechanically strong (through tensile test) 3D multilayer device was obtained using plasma treatment with oxygen and methane as precursors followed by pressing at temperature near of Tg + 20 of the polymer with the lowest Tg (PS). This nanotexturing technique is also used to facilitate thermal and mechanical stability of the microchannels for microfluidic applications. X-ray tomography measurements showed that 3D polymer made chips, at certain plasma and press bonding conditions, have structural integrity and no deformation were detected in channels shape post thermal pressing process. The dimension stability of channels and reservoirs and the rigid interfacial region at PEEK-PS make this chip design attractive and feasible for advanced lab-on-a-chip applications.

Three Dimensional Honeycomb Patterned Fibrinogen Based Nanofibers Induce Substantial Osteogenic Response of Mesenchymal Stem Cells.

Stem cells therapy offers a viable alternative for treatment of bone disorders to the conventional bone grafting. However clinical therapies are still hindered by the insufficient knowledge on the conditions that maximize stem cells differentiation. Hereby, we introduce a novel 3D honeycomb architecture scaffold that strongly support osteogenic differentiation of human adipose derived mesenchymal stem cells (ADMSCs). The scaffold is based on electrospun hybrid nanofibers consisting of poly (L-lactide ε-caprolactone) and fibrinogen (PLCL/FBG). Classical fibers orientations, random or aligned were also produced and studied for comparison. The overall morphology of ADMSC's generally followed the nanofibers orientation and dimensionality developing regular focal adhesions and direction-dependent actin cytoskeleton bundles. However, there was an initial tendency for cells rounding on honeycomb scaffolds before ADMSCs formed a distinct bridging network. This specific cells organization appeared to have significant impact on the differentiation potential of ADMSCs towards osteogenic lineage, as indicated by the alkaline phosphatase production, calcium deposition and specific genes expression. Collectively, it was observed synergistic effect of nanofibers with honeycomb architecture on the behavior of ADMSCs entering osteogenic path of differentiation which outlines the potential benefits from insertion of such bioinspired geometrical cues within scaffolds for bone tissue engineering.

Dynamic adhesive environment alters the differentiation potential of young and ageing mesenchymal stem cells.

Engineering dynamic stem cell niche-like environment offers opportunity to obtain better control of the fate of stem cells. We identified, for the first time, that periodic changes in the adhesive environment of human adipose derived mesenchymal stem cells (ADSCs) alters dramatically their asymmetric division but not their ability for symmetric renewal. Hereby, we used smart thermo-responsive polymer (PNIPAM) to create a dynamic adhesive environment for ADSCs by applying periodic temperature cycles to perturb adsorbed adhesive proteins to substratum interaction. Cumulative population doubling time (CPDT) curves showed insignificant decline in the symmetric cell growth studied for up to 13th passages accompanied with small changes in the overall cell morphology and moderately declined fibronectin (FN) matrix deposition probably as a functional consequence of ADSCs ageing. However, a substantial alteration in the differentiation potential of ADSCs from both early and late passages (3rd and 14th, respectively) was found when the cells were switched to osteogenic differentiation conditions. This behavior was evidenced by the significantly altered alkaline phosphatase activity and Ca deposition (Alizarin red) assayed at 3, 14 and 21day in comparison to the control samples of regular TC polystyrene processed under same temperature settings.

Functionalized, biocompatible, and impermeable nanoscale coatings for PEEK.

Biologically compatible coatings that provide hermetic seal could resolve a major technological hurdle in the attempt to replace metals with polymers for biochips and active medical implants. The use of amorphous carbon/diamond like carbon (a-C:H) coatings to hermetically seal and biologically enhance polyether-ether-ketone (PEEK) for biomedical device integration in the human body was investigated. The PEEK coating functionality (sp3/sp2 ratio), hardness and thickness (70-200nm) were controlled, by varying H2 and N2 concentration during the plasma operation with CH4. a-C:H coatings having the highest indentation modulus of 13.5GPa, originate out of a CH4 (90%) rich composition. Even in a mixture of 70/30 H2/CH4 the hardness is 4.76GPa, corresponding to hard and dense coatings. In all tested conditions of deposition coatings hardens was sufficient for the purpose of PEEK implants modification. The synthesized (a-C:H) nanoscale coatings were not water permeable as measured by the hydrolysis test, resolving the traditional challenge of swelling in wet environment. The hardness of the coatings showed strong correlations with the thickness, surprisingly however, with no correlations with the sp3/sp2 ratio. Selected non water permeable nanoscale coating on PEEK showed strong bioactivity by being viable for human osteoblast (hFOB) and human fibroblast (hGF) cells without toxicity issues. No correlation was observed between the coatings sp3/sp2 ratio and biological performance.

Osteogenic differentiation of mesenchymal stem cells using hybrid nanofibers with different configurations and dimensionality.

Novel, hybrid fibrinogen/polylactic acid (FBG/PLA) nanofibers with different configuration (random vs aligned) and dimensionality (2-D vs 3-D environment) were used to control the overall behavior and the osteogenic differentiation of human adipose-derived mesenchymal stem cells (ADMSCs). Aligned nanofibers in both the 2-D and 3-D configurations are proved to be favored for osteodifferentiation. Morphologically, we found that on randomly configured nanofibers, the cells developed a stellate-like morphology with multiple projections; however, time-lapse analysis showed significantly diminished cell movements. Conversely, an elongated cell shape with advanced cell spreading and extended actin cytoskeleton accompanied with significantly increased cell mobility were observed when cells attached on aligned nanofibers. Moreover, a clear tendency for higher alkaline phosphatase activity was also found on aligned fibers when ADMSCs were switched to osteogenic induction medium. The strongest accumulation of Alizarin red (AR) and von Kossa stain at 21 days of culture in osteogenic medium were found on 3-D aligned constructs while the rest showed lower and rather undistinguishable activity. Quantitative reverse transcription-polymerase chain reaction analysis for Osteopontin (OSP) and RUNX 2 generally confirmed this trend showing favorable expression of osteogenic genes activity in 3-D environment particularly in aligned configuration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2065-2074, 2017.

Differentiation of Human Mesenchymal Stem Cells Toward Quality Cartilage Using Fibrinogen-Based Nanofibers.

Mimicking the complex intricacies of the extra cellular matrix including 3D configurations and aligned fibrous structures were traditionally perused for producing cartilage tissue from stem cells. This study shows that human adipose derived mesenchymal stem cells (hADMSCs) establishes significant chondrogenic differentiation and may generate quality cartilage when cultured on 2D and randomly oriented fibrinogen/poly-lactic acid nanofibers compared to 3D sandwich-like environments. The adhering cells show well-developed focal adhesion complexes and actin cytoskeleton arrangements confirming the proper cellular interaction with either random or aligned nanofibers. However, quantitative reverse transcription-polymerase chain reaction analysis for Collagen 2 and Collagen 10 genes expression confirms favorable chondrogenic response of hADMSCs on random nanofibers and shows substantially higher efficacy of their differentiation in 2D configuration versus 3D constructs. These findings introduce a new direction for cartilage tissue engineering through providing a simple platform for the routine generation of transplantable stem cells derived articular cartilage replacement that might improve joint function.

Tribological characteristics of few-layer graphene over Ni grain and interface boundaries.

The tribological properties of metal-supported few-layered graphene depend strongly on the grain topology of the metal substrate. Inhomogeneous distribution of graphene layers at such regions led to variable landscapes with distinguishable roughness. This discrepancy in morphology significantly affects the frictional and wetting characteristics of the FLG system. We discretely measured friction characteristics of FLG covering grains and interfacial grain boundaries of polycrystalline Ni metal substrate via an atomic force microscopy (AFM) probe. The friction coefficient of FLG covered at interfacial grain boundaries is found to be lower than that on grains in vacuum (at 10(-5) Torr pressure) and similar results were obtained in air condition. Sliding history with AFM cantilever, static and dynamic pull-in and pull-off adhesion forces were addressed in the course of friction measurements to explain the role of the out-of-plane deformation of graphene layer(s). Finite element simulations showed good agreement with experiments and led to a rationalization of the observations. Thus, with interfacial grain boundaries the FLG tribology can be effectively tuned.

Exploring molecular changes at the surface of polypropylene after accelerated thermomolecular adhesion treatments.

A central composite rotatable design (CCRD) method was used to investigate the performance of the accelerated thermomolecular adhesion process (ATmaP), at different operating conditions. ATmaP is a modified flame-treatment process that features the injection of a coupling agent into the flame to impart a tailored molecular surface chemistry on the work piece. In this study, the surface properties of treated polypropylene were evaluated using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). All samples showed a significant increase in the relative concentration of oxygen (up to 12.2%) and nitrogen (up to 2.4%) at the surface in comparison with the untreated sample (0.7% oxygen and no detectable nitrogen) as measured by XPS. ToF-SIMS and principal components analysis (PCA) showed that ATmaP induced multiple reactions at the polypropylene surface such as chain scission, oxidation, nitration, condensation, and molecular loss, as indicated by changes in the relative intensities of the hydrocarbon (C(3)H(7)(+), C(3)H(5)(+), C(4)H(7)(+), and C(5)H(9)(+)), nitrogen and oxygen-containing secondary ions (C(2)H(3)O(+), C(3)H(8)N(+), C(2)H(5)NO(+), C(3)H(6)NO(+), and C(3)H(7)NO(+)). The increase in relative intensity of the nitrogen oxide ions (C(2)H(5)NO(+) and C(3)H(7)NO(+)) correlates with the process of incorporating oxides of nitrogen into the surface as a result of the injection of the ATmaP coupling agent.