Genetic profiling of human bone marrow and adipose tissue-derived mesenchymal stem cells reveals differences in osteogenic signaling mediated by graphene.

BACKGROUND: In the last decade, graphene surfaces have consistently supported osteoblast development of stem cells, holding promise as a therapeutic implant for degenerative bone diseases. However, until now no study has specifically examined the genetic changes when stem cells undergo osteogenic differentiation on graphene. RESULTS: In this study, we provide a detailed overview of gene expressions when human mesenchymal stem cells (MSCs) derived from either adipose tissue (AD-MSCs) or bone marrow (BM-MSCs), are cultured on graphene. Genetic expressions were measured using osteogenic RT2 profiler PCR arrays and compared either over time (7 or 21 days) or between each cell source at each time point. Genes were categorized as either transcriptional regulation, osteoblast-related, extracellular matrix, cellular adhesion, BMP and SMAD signaling, growth factors, or angiogenic factors. Results showed that both MSC sources cultured on low oxygen graphene surfaces achieved osteogenesis by 21 days and expressed specific osteoblast markers. However, each MSC source cultured on graphene did have genetically different responses. When compared between each other, we found that genes of BM-MSCs were robustly expressed, and more noticeable after 7 days of culturing, suggesting BM-MSCs initiate osteogenesis at an earlier time point than AD-MSCs on graphene. Additionally, we found upregulated angiogenic markers in both MSCs sources, suggesting graphene could simultaneously attract the ingrowth of blood vessels in vivo. Finally, we identified several novel targets, including distal-less homeobox 5 (DLX5) and phosphate-regulating endopeptidase homolog, X-linked (PHEX). CONCLUSIONS: Overall, this study shows that graphene genetically supports differentiation of both AD-MSCs and BM-MSCs but may involve different signaling mechanisms to achieve osteogenesis. Data further demonstrates the lack of aberrant signaling due to cell-graphene interaction, strengthening the application of specific form and concentration of graphene nanoparticles in bone tissue engineering.

Dendritic cell biocompatibility of ether-based urethane films.

The use of synthetic materials for biomedical applications is ever expanding. One of the major requirements for these materials is biocompatibility, which includes prevention of immune system responses. Due to the inherent complexity of their structural composition, the polyurethane (PU) family of polymers is being used in a variety of medical applications, from soft and hard tissue scaffolds to intricate coatings on implantable devices. Herein, we investigated whether two polymer materials, D3 and D7, induced an immune response, measured by their effects on a dendritic cell (DC) line, JAWS II. Using a lactate dehydrogenase cytotoxicity assay and Annexin V/PI staining, we found that the PU materials did not induce cytotoxicity in DC cells. Using confocal microscopy, we also showed that the materials did not induce activation or maturation, as compared to positive controls. This was confirmed by looking at various markers, CD80, CD86, MHC class I, and MHC class II, via flow cytometry. Overall, the results indicated that the investigated PU films are biocompatible in terms of immunotoxicology and immunogenicity and show great promise for use in regenerative medicine.

Benign zinc oxide betaine-modified biochar nanocomposites for phosphate removal from aqueous solutions.

Phosphate is one of the most costly and complex environmental pollutants that leads to eutrophication, which decreases water quality and access to clean water. Among different adsorbents, biochar is one of the promising adsorbents for phosphate removal as well as heavy metal removal from an aqueous solution. In this study, biochar was impregnated with nano zinc oxide in the presence of glycine betaine. The Zinc Oxide Betaine-Modified Biochar Nanocomposites (ZnOBBNC) proved to be an excellent adsorbent for the removal of phosphate, exhibiting a maximum adsorption capacity of phosphate (265.5 mg. g-1) and fast adsorption kinetics (~100% removal at 15 min at 10 mg. L-1 phosphate and 3 g. L-1 nanocomposite dosage) in phosphate solution. The synthesis of these benign ZnOBBNC involves a process that is eco-friendly and economically feasible. From material characterization, we found that the ZnOBBNC has ~20-30 nm particle size, high surface area (100.01 m2. g-1), microporous (25.79 Å) structures, and 7.64% zinc content. The influence of pH (2-10), coexisting anions (Cl-, CO32-, NO3- and SO43-), initial phosphate concentration (10-500 mg. L-1), and ZnOBBNC dosage (0.5-5 g. L-1) were investigated in batch experiments. From the adsorption isotherms data, the adsorption of phosphate using ZnOBBNC followed Langmuir isotherm (R2 = 0.9616), confirming the mono-layered adsorption mechanism. The kinetic studies showed that the phosphate adsorption using ZnOBBNC followed the pseudo-second-order model (R2 = 1.0000), confirming the chemisorption adsorption mechanism with inner-sphere complexion. Our results demonstrated ZnOBBNC as a suitable, competitive candidate for phosphate removal from both mock lab-prepared and real field-collected wastewater samples when compared to commercial nanocomposites.

Cytotoxicity profile of pristine graphene on brain microvascular endothelial cells.

Graphene-based nanomaterials hold the potential to be used in a wide variety of applications, including biomedical devices. Pristine graphene (PG) is an un-functionalized, defect-free type of graphene that could be used as a material for neural interfacing. However, the neurotoxic effects of PG, particularly to the blood-brain barrier (BBB), have not been fully studied. The BBB separates the brain tissue from the circulating substances in the blood and is essential to maintain the brain homeostasis. The principal components of the BBB are brain microvascular endothelial cells (BMVECs), which maintain a protectively low permeability due to the expression of tight junction proteins. Here we analyzed the effects of PG on BMVECs in an in vitro model of the BBB. BMVECs were treated with PG at 0, 10, 50 and 100 µg/mL for 24 hours and viability and functional analyses of BBB integrity were performed. PG increased lactate dehydrogenase release at 50 and 100 µg/mL, suggesting the induction of necrosis. Surprisingly, 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium (XTT) conversion was increased at 10 and 50 µg/mL. In contrast, XTT conversion was decreased at 100 µg/mL, suggesting the induction of cell death. In addition, 100 µg/mL PG increased DNA fragmentation, suggesting induction of apoptosis. At the same time, 50 and 100 µg/mL of PG increased the endothelial permeability, which corresponded with a decrease in the expression of the tight junction protein occludin at 100 µg/mL. In conclusion, these results suggest that PG negatively affects the viability and function of the BBB endothelial cells in vitro.

In vivo noninvasive analysis of graphene nanomaterial pharmacokinetics using photoacoustic flow cytometry.

Graphene-based nanomaterials (GBNs) are quickly revolutionizing modern electronics, energy generation and storage, clothing and biomedical devices. Due to GBN's variety of physical and chemical parameters that define their toxicity and their aggregation in suspension, interpreting its toxicology without accurate information on graphene's distribution and behavior in live organisms is challenging. In this work, we present a laser-based optical detection methodology for noninvasive detection and pharmacokinetics analysis of GBNs directly in blood flow in mice using in vivo photoacoustic (PA) flow cytometry (PAFC). PAFC provides unique insight on how chemical modifications of GBNs affect their distribution in blood circulation and how quickly they are eliminated from the flow. Overall, PAFC provided unique data crucial for understanding GBN toxicity through real-time detection of GBNs using their intrinsic light absorption contrast. Copyright © 2017 John Wiley & Sons, Ltd.

Physicochemical characteristics of pristine and functionalized graphene.

Graphene-based nanomaterials have received significant attention in the last decade due to their interesting properties. Its electrical and thermal conductivity and strength make graphene well suited for a variety of applications, particularly for use as a composite material in plastics. Furthermore, much work is taking place to utilize graphene as a biomaterial for uses such as drug delivery and tissue regeneration scaffolds. Owing to the rapid progress of graphene and its potential in many marketplaces, the potential toxicity of these materials has garnered attention. Graphene, while simple in its purest form, can have many different chemical and physical properties. In this paper, we describe our toxicity evaluation of pristine graphene and a functionalized graphene sample that has been oxidized for enhanced hydrophilicity, which was synthesized from the pristine sample. The samples were characterized by X-ray photoelectron spectroscopy, Raman spectroscopy, infrared spectroscopy, thermogravimetric analysis, zeta-potential, atomic force microscopy and electron microscopy. We discuss the disagreement between the size of imaged samples analyzed by atomic force microscopy and by transmission electron microscopy. Furthermore, the samples each exhibit quite different surface chemistry and structure, which directly affects their interaction with aqueous environments and is important to consider when evaluating the toxicity of materials both in vitro and in vivo. Copyright © 2017 John Wiley & Sons, Ltd.

The role of surface chemistry in the cytotoxicity profile of graphene.

Graphene and its derivative, because of their unique physical, electrical and chemical properties, are an important class of nanomaterials being proposed as foundational materials in nanomedicine as well as for a variety of industrial applications. A major limitation for graphene, when used in biomedical applications, is its poor solubility due to its rather hydrophobic nature. Therefore, chemical functionalities are commonly introduced to alter both its surface chemistry and biochemical activity. Here, we show that surface chemistry plays a major role in the toxicological profile of the graphene structures. To demonstrate this, we chemically increased the oxidation level of the pristine graphene and compared the corresponding toxicological effects along with those for the graphene oxide. X-ray photoelectron spectroscopy revealed that pristine graphene had the lowest amount of surface oxygen, while graphene oxide had the highest at 2.5% and 31%, respectively. Low and high oxygen functionalized graphene samples were found to have 6.6% and 24% surface oxygen, respectively. Our results showed a dose-dependent trend in the cytotoxicity profile, where pristine graphene was the most cytotoxic, with decreasing toxicity observed with increasing oxygen content. Increased surface oxygen also played a role in nanomaterial dispersion in water or cell culture medium over longer periods. It is likely that higher dispersity might result in graphene entering into cells as individual flakes ~1 nm thick rather than as more cytotoxic aggregates. In conclusion, changes in graphene's surface chemistry resulted in altered solubility and toxicity, suggesting that a generalized toxicity profile would be rather misleading. Copyright © 2016 John Wiley & Sons, Ltd.

p53-competent cells and p53-deficient cells display different susceptibility to oxygen functionalized graphene cytotoxicity and genotoxicity.

Due to the distinctive physical, electrical, and chemical properties of graphene nanomaterials, numerous efforts pursuing graphene-based biomedical and industrial applications are underway. Oxidation of pristine graphene surfaces mitigates its otherwise hydrophobic characteristic thereby improving its biocompatibility and functionality. Yet, the potential widespread use of oxidized graphene derivatives raises concern about adverse impacts on human health. The p53 tumor suppressor protein maintains cellular and genetic stability after toxic exposures. Here, we show that p53 functional status correlates with oxygen functionalized graphene (f-G) cytotoxicity and genotoxicity in vitro. The f-G exposed p53-competent cells, but not p53-deficient cells, initiated G0 /G1 phase cell cycle arrest, suppressed reactive oxygen species, and entered apoptosis. There was p53-dependent f-G genotoxicity evident as increased structural chromosome damage, but not increased gene mutation or chromatin loss. In conclusion, the cytotoxic and genotoxic potential for f-G in exposed cells was dependent on the p53 functional status. These findings have broad implications for the safe and effective implementation of oxidized graphene derivatives into biomedical and industrial applications. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Graphene nanoparticles as osteoinductive and osteoconductive platform for stem cell and bone regeneration.

The potential of graphene-based nanoparticles (GNPs) has recently gained significant attention in biomedicine, especially in tissue engineering. In this study, we investigated the osteoinductive and osteoconductive effects of low oxygen content graphene (LOG) nanoparticles on adult mesenchymal stem cells (MSCs) in vitro and in vivo. We showed that adult goat MSCs were viable in the presence of 0.1 mg/mL LOG and retained their stem cell properties. A 3D scaffold made from agarose was used to encapsulate MSCs and LOG nanoparticles. Scanning electron microscopy demonstrated the cell morphology and adherence of MSCs to LOG in the 3D form. The LOG and MSCs in the 3D scaffold were xenogenically implanted into a rat unicortical tibial bone defect. The combination of MSCs and LOG nanoparticles resulted in improved active bone formation and increased mineralization. These results strengthen the applicability of LOG nanoparticles as an adjunct treatment for bone tissue engineering.

Graphene supports in vitro proliferation and osteogenic differentiation of goat adult mesenchymal stem cells: potential for bone tissue engineering.

Current treatments for bone loss injuries involve autologous and allogenic bone grafts, metal alloys and ceramics. Although these therapies have proved useful, they suffer from inherent challenges, and hence, an adequate bone replacement therapy has not yet been found. We hypothesize that graphene may be a useful nanoscaffold for mesenchymal stem cells and will promote proliferation and differentiation into bone progenitor cells. In this study, we evaluate graphene, a biocompatible inert nanomaterial, for its effect on in vitro growth and differentiation of goat adult mesenchymal stem cells. Cell proliferation and differentiation are compared between polystyrene-coated tissue culture plates and graphene-coated plates. Graphitic materials are cytocompatible and support cell adhesion and proliferation. Importantly, cells seeded on to oxidized graphene films undergo osteogenic differentiation in fetal bovine serum-containing medium without the addition of any glucocorticoid or specific growth factors. These findings support graphene's potential to act as an osteoinducer and a vehicle to deliver mesenchymal stem cells, and suggest that the combination of graphene and goat mesenchymal stem cells provides a promising construct for bone tissue engineering.

Ammonia gas sensing behavior of tanninsulfonic acid doped polyaniline-TiO2 composite.

A highly active tannin doped polyaniline-TiO2 composite ammonia gas sensor was developed and the mechanism behind the gas sensing activity was reported for the first time. A tanninsulfonic acid doped polyaniline (TANIPANI)-titanium dioxide nanocomposite was synthesized by an in situ polymerization of aniline in the presence of tanninsulfonic acid and titanium dioxide nanoparticles. X-ray diffraction and thermogravimetric analysis were utilized to determine the incorporation of TiO2 in TANIPANI matrix. UV-Visible and infrared spectroscopy studies provided information about the electronic interactions among tannin, polyaniline, and TiO2. Scanning electron microscopy (SEM) along with energy dispersive X-ray spectroscopy (EDS) and atomic force microscopy (AFM) surface analysis techniques were used to investigate the metal oxide dispersions inside polyaniline matrix. Gas sensors were prepared by spin coating solutions of TANIPANI-TiO2 and TANIPANI composites onto glass slides. Sensors were tested at three different concentrations (20 ppm, 40 ppm, and 60 ppm) of ammonia gas at ambient temperature conditions by measuring the changes in surface resistivity of the films with respect to time. Ammonia gas sensing plots are presented showing the response values, response times and recovery times. The TANIPANI-TiO2 composite exhibited better response and shorter recovery times when compared to TANIPANI control and other polyaniline composites that have been reported in the literature. For the first time a proposed mechanism of gas sensing basing on the polaron band localization and its effects on the gas sensing behavior of polyaniline are reported.

Xenogenic Implantation of Human Mesenchymal Stromal Cells Using a Novel 3D-Printed Scaffold of PLGA and Graphene Leads to a Significant Increase in Bone Mineralization in a Rat Segmental Femoral Bone Defect.

Tissue-engineering technologies have the potential to provide an effective approach to bone regeneration. Based on the published literature and data from our laboratory, two biomaterial inks containing PLGA and blended with graphene nanoparticles were fabricated. The biomaterial inks consisted of two forms of commercially available PLGA with varying ratios of LA:GA (65:35 and 75:25) and molecular weights of 30,000-107,000. Each of these forms of PLGA was blended with a form containing a 50:50 ratio of LA:GA, resulting in ratios of 50:65 and 50:75, which were subsequently mixed with a 0.05 wt% low-oxygen-functionalized derivative of graphene. Scanning electron microscopy showed interconnected pores in the lattice structures of each scaffold. The cytocompatibility of human ADMSCs transduced with a red fluorescent protein (RFP) was evaluated in vitro. The in vivo biocompatibility and the potential to repair bones were evaluated in a critically sized 5 mm mechanical load-bearing segmental femur defect model in rats. Bone repair was monitored by radiological, histological, and microcomputed tomography methods. The results showed that all of the constructs were biocompatible and did not exhibit any adverse effects. The constructs containing PLGA (50:75)/graphene alone and with hADMSCs demonstrated a significant increase in mineralized tissues within 60 days post-treatment. The percentage of bone volume to total volume from microCT analyses in the rats treated with the PLGA + cells construct showed a 50% new tissue formation, which matched that of a phantom. The microCT results were supported by Von Kossa staining.

Synthesis of "Naked" TeO2 Nanoparticles for Biomedical Applications.

Chalcogenide nanoparticles have become a very active field of research for their optoelectronic and biological properties. This article shows the production of tellurium dioxide nanoparticles (TeO2 NPs) by pulsed laser ablation in liquids. The produced nanoparticles were spherical with a diameter of around 70 nm. The energy band gap of those nanoparticles was determined to be around 5.2 eV. Moreover, TeO2 NPs displayed a dose-dependent antibacterial effect against antibiotic-resistant bacteria such as multidrug-resistant Escherichia coli (MDR E. coli) and methicillin-resistant Staphylococcus aureus (MR S. aureus). The "naked" nature of the nanoparticle surface helped to eradicate the antibiotic-resistant bacteria at a very low concentration, with IC50 values of ∼4.3 ± 0.9 and 3.7 ± 0.2 ppm for MDR E. coli and MR S. aureus, respectively, after just 8 h of culture. Further, the IC50 values of the naked TeO2 NPs against melanoma (skin cancer) and healthy fibroblasts were 1.6 ± 0.7 and 5.5 ± 0.2 ppm, respectively, for up to 72 h. Finally, to understand these optimal antibacterial and anticancer properties of the TeO2 NPs, the reactive oxygen species generated by the nanoparticles were measured. In summary, the present in vitro results demonstrate much promise for the presently prepared TeO2 NPs and they should be studied for a wide range of safe antibacterial and anticancer applications.

Exceptional superhydrophobicity and low velocity impact icephobicity of acetone-functionalized carbon nanotube films.

We present a simple method to produce carbon nanotube-based films with exceptional superhydrophobicity and impact icephobicity by depositing acetone-treated single-walled carbon nanotubes on glass substrates. This method is scalable and can be adopted for any substrate, both flexible and rigid. These films have indicated a high contact angle, in the vicinity of 170°, proved both by static and dynamic analysis processes. The dynamic evaporation studies indicated that a droplet deposited on the treated films evaporated in the constant contact angle mode for more than 80% of the total evaporation time, which is definitely a characteristic of superhydrophobic surfaces. Furthermore, the acetone-functionalized films showed a strong ability to mitigate ice accretion from supercooled water droplets (-8 °C), when the droplets were found to bounce off the films tilted at 30°. The untreated nanotube films did not indicate similar behavior, and the supercooled water droplets remained attached to the films' surfaces. Such studies could be the foundation of highly versatile technologies for both water and ice mitigation.

Evaluation of a bone filler scaffold for local antibiotic delivery to prevent Staphylococcus aureus infection in a contaminated bone defect.

We previously reported the development of an osteogenic bone filler scaffold consisting of degradable polyurethane, hydroxyapatite, and decellularized bovine bone particles. The current study was aimed at evaluating the use of this scaffold as a means of local antibiotic delivery to prevent infection in a bone defect contaminated with Staphylococcus aureus. We evaluated two scaffold formulations with the same component ratios but differing overall porosity and surface area. Studies with vancomycin, daptomycin, and gentamicin confirmed that antibiotic uptake was concentration dependent and that increased porosity correlated with increased uptake and prolonged antibiotic release. We also demonstrate that vancomycin can be passively loaded into either formulation in sufficient concentration to prevent infection in a rabbit model of a contaminated segmental bone defect. Moreover, even in those few cases in which complete eradication was not achieved, the number of viable bacteria in the bone was significantly reduced by treatment and there was no radiographic evidence of osteomyelitis. Radiographs and microcomputed tomography (µCT) analysis from the in vivo studies also suggested that the addition of vancomycin did not have any significant effect on the scaffold itself. These results demonstrate the potential utility of our bone regeneration scaffold for local antibiotic delivery to prevent infection in contaminated bone defects.

Phosphate removal from wastewater using novel renewable resource-based, cerium/manganese oxide-based nanocomposites.

Nanocomposites containing mixed metal oxides show excellent phosphate removal results and are better compared to individual metal oxides. In this research, cerium/manganese oxide nanocomposites, embedded on the surface of modified cellulose pine wood shaving, were synthesized by a simple technique that is both eco-friendly and economically feasible. No toxic or petroleum chemicals were employed during preparation. Scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), surface area analysis, and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy were performed to study the shape and size of nanocomposites as well as composition of elements present on the surface of the nanocomposites. Adsorption isotherm (Langmuir, Freundlich, Temkin, and Dubinin-Radushkevich) and kinetic studies (pseudo first and second-order, Elovich and Weber-Morris) were carried out to determine the adsorption mechanism for phosphate removal from contaminated water. The maximum adsorption capacity of nanocomposites was found to be 204.09 mg/g, 174.42 mg/g, and 249.33 mg/g for 100 mg, 300 mg, and 500 mg, respectively. The results indicate that the nanocomposites were able to decrease the phosphorus concentration from 10 to 0.01 ppm, below the threshold limit required by EPA guidelines in the USA. We also demonstrated that the media could be regenerated and reused five times without loss of performance.

Graphene-based 2D constructs for enhanced fibroblast support.

Complex skin wounds have always been a significant health and economic problem worldwide due to their elusive and sometimes poor or non-healing conditions. If not well-treated, such wounds may lead to amputation, infections, cancer, or even death. Thus, there is a need to efficiently generate multifunctional skin grafts that address a wide range of skin conditions, including non-healing wounds, and enable the regeneration of new skin tissue. Here, we propose studying pristine graphene and two of its oxygen-functionalized derivatives-high and low-oxygen graphene films-as potential substrates for skin cell proliferation and differentiation. Using BJ cells (human foreskin-derived fibroblasts) to represent basic skin cells, we show that the changes in surface properties of pristine graphene due to oxygen functionalization do not seem to statistically impact the normal proliferation and maturation of skin cells. Our results indicate that the pristine and oxidized graphenes presented relatively low cytotoxicity to BJ fibroblasts and, in fact, support their growth and bioactivity. Therefore, these graphene films could potentially be integrated into more complex skin regenerative systems to support skin regeneration. Because graphene's surface can be relatively easily functionalized with various chemical groups, this finding presents a major opportunity for the development of various composite materials that can act as active components in regenerative applications such as skin regeneration.

Optimizing Lignosulfonic Acid-Grafted Polyaniline as a Hole-Transport Layer for Inverted CH3NH3PbI3 Perovskite Solar Cells.

A conducting polymer of lignosulfonic acid-grafted, polyaniline-doped camphorsulfonic acid (LS-PANI-CSA), created via a low-temperature solution process, has been explored as an efficient hole-transport layer (HTL) for inverted single cation-anion CH3NH3PbI3 perovskite solar cells. The performance of the solar cell was optimized in this study by tuning the morphology and work function of LS-PANI-CSA films using dimethylsulfoxide (DMSO) as a solvent in treatment. Results showed that DMSO washing enhanced the electronic properties of the LS-PANI-CSA film and increased its hydrophobicity, which is very important for perovskite growth. The perovskite active layer deposited onto the DMSO-treated LS-PANI-CSA layer had higher crystallinity with large grain sizes (>5 µm), more uniform and complete surface coverage, and very low pinhole density and PbI2 residues compared to untreated LS-PANI-CSA. These enhancements result in higher device performance and stability. Using DMSO-treated LS-PANI-CSA as an HTL at 15 nm of thickness, a maximum 10.8% power conversion efficiency was obtained in ITO/LS-PANI-CSA/MAPbI3/PCBM/BCP/Ag inverted-device configurations. This was a significant improvement compared to 5.18% for devices based on untreated LS-PANI-CSA and a slight improvement over PEDOT:PSS-based devices with 9.48%. Furthermore, the perovskite based on treated LS-PANI-CSA showed the higher stability compared to both untreated LS-PANI-CSA and PEDOT:PSS HTL-based devices.

Functionalized Graphene Nanoparticles Induce Human Mesenchymal Stem Cells to Express Distinct Extracellular Matrix Proteins Mediating Osteogenesis.

PURPOSE: The extracellular matrix (ECM) labyrinthine network secreted by mesenchymal stem cells (MSCs) provides a microenvironment that enhances cell adherence, proliferation, viability, and differentiation. The potential of graphene-based nanomaterials to mimic a tissue-specific ECM has been recognized in designing bone tissue engineering scaffolds. In this study, we investigated the expression of specific ECM proteins when human fat-derived adult MSCs adhered and underwent osteogenic differentiation in the presence of functionalized graphene nanoparticles. METHODS: Graphene nanoparticles with 6-10% oxygen content were prepared and characterized by XPS, FTIR, AFM and Raman spectroscopy. Calcein-am and crystal violet staining were performed to evaluate viability and proliferation of human fat-derived MSCs on graphene nanoparticles. Alizarin red staining and quantitation were used to determine the effect of graphene nanoparticles on osteogenic differentiation. Finally, immunofluorescence assays were used to investigate the expression of ECM proteins during cell adhesion and osteogenic differentiation. RESULTS: Our data show that in the presence of graphene, MSCs express specific integrin heterodimers and exhibit a distinct pattern of the corresponding bone-specific ECM proteins, primarily fibronectin, collagen I and vitronectin. Furthermore, MSCs undergo osteogenic differentiation spontaneously without any chemical induction, suggesting that the physicochemical properties of graphene nanoparticles might trigger the expression of bone-specific ECM. CONCLUSION: Understanding the cell-graphene interactions resulting in an osteogenic niche for MSCs will significantly improve the application of graphene nanoparticles in bone repair and regeneration.

Multiomics Evaluation of Human Fat-Derived Mesenchymal Stem Cells on an Osteobiologic Nanocomposite.

Effective graft technologies for bone repair have been a primary focus in the field of bone tissue engineering. We have previously fabricated and examined a nanocomposite composed of polyurethane, nano-hydroxyapatite, and decellularized bone particles, which demonstrated osteobiologic characteristics. To evaluate the underlying mechanisms of this biomaterial, human adipose-derived mesenchymal stem cell seeded scaffolds were assessed using a combinatorial approach of transcriptomic and metabolomic analyses. Data from osteogenic and signal transduction polymerase chain reaction arrays and small molecule abundances, measured through liquid chromatography-mass spectrometry, were cross-examined using Integrated Molecular Pathway Level Analysis, Database for Annotation, Visualization, and Integrated Discovery, and ConsensusPathDB online tools to generate a fundamental collection of scaffold-influenced pathways. Results demonstrated upregulation of key osteogenic, cellular adhesion cell signaling markers and indicated that Hedgehog and Wnt signaling pathways were primary candidates for the osteobiologic mechanisms of the scaffold design. The detection of complimentary metabolites, such as ascorbate, further indicates that scaffolds generate intricate cellular environments, promoting cell attachment and subsequent osteodifferentiation.