Nileshkumar Dubey

Graphene: A Versatile Carbon-Based Material for Bone Tissue Engineering

The development of materials and strategies that can influence stem cell attachment, proliferation, and differentiation towards osteoblasts is of high interest to promote faster healing and reconstructions of large bone defects. Graphene and its derivatives (graphene oxide and reduced graphene oxide) have received increasing attention for biomedical applications as they present remarkable properties such as high surface area, high mechanical strength, and ease of functionalization. These biocompatible carbon-based materials can induce and sustain stem cell growth and differentiation into various lineages. Furthermore, graphene has the ability to promote and enhance osteogenic differentiation making it an interesting material for bone regeneration research. This paper will review the important advances in the ability of graphene and its related forms to induce stem cells differentiation into osteogenic lineages.

Graphene oxide-based substrate: physical and surface characterization, cytocompatibility and differentiation potential of dental pulp stem cells

OBJECTIVE The aim of this study was to evaluate the cytotoxicity and differentiation potential of a graphene oxide (GO)-based substrate using dental pulp stem cell (DPSC). METHODS GO was obtained via chemical exfoliation of graphite using the modified Hummers method and dispersed in water-methanol solution. 250μL of 1.5mg/mL solution were added to a cover slip and allowed to dry (25°C, 24h). GO-based substrate was characterized by Raman spectroscopy, AFM and contact angle. DPSC were seeded on GO and glass (control). Cell attachment and proliferation were evaluated by polymeric F-actin staining, SEM and MTS assay for five days. mRNA expression of MSX-1, PAX-9, RUNX2, COL I, DMP-1 and DSPP were evaluated by qPCR (7 and 14 days). Statistical analyses were performed by either Mann-Whitney, one or two-way Anova followed by and Tukeys post hoc analysis (α=0.05). RESULTS Peaks at 1587cm(-1) and 1340cm(-1) (G and D band) and ID/IG of 0.83 were observed for GO with Raman. AFM showed that GO was randomly deposited and created a rougher surface comparing to the control. Cells successfully adhered on both substrates. There was no difference in cell proliferation after 5 days. Cells on GO presented higher expression for all genes tested except MSX-1 and RUNX2 for 7 days. SIGNIFICANCE GO-based substrate allowed DPSC attachment, proliferation and increased the expression of several genes that are upregulated in mineral-producing cells. These findings open opportunities to the use of GO alone or in combination with dental materials to improve their bioactivity and beyond.

Bioactivity, physical and chemical properties of MTA mixed with propylene glycol

Objective To investigate the physical (setting time, hardness, flowability, microstructure) and chemical (pH change, calcium release, crystallinity) properties and the biological outcomes (cell survival and differentiation) of mineral trioxide aggregate (MTA) mixed using different proportions of propylene glycol (PG) and water. Material and Methods White MTA was mixed with different water/PG ratios (100/0, 80/20 and 50/50). Composition (XRD), microstructure (SEM), setting time (ASTM C266-13), flowability (ANSI/ADA 57-2000), Knoop hardness (100 g/10 s) and chemical characteristics (pH change and Ca2+ release for 7 days) were evaluated. Cell proliferation, osteo/odontoblastic gene expression and mineralization induced by MTA mixed with PG were evaluated. MTA discs (5 mm in diameter, 2 mm thick) were prepared and soaked in culture medium for 7 days. Next, the discs were removed and the medium used to culture dental pulp stem cells (DPSC) for 28 days. Cells survival was evaluated using MTS assay (24, 72 and 120 h) and differentiation with RT-PCR (ALP, OCN, Runx2, DSPP and MEPE) and alizarin red staining (7 and 14 days). Data were analysed using one-way ANOVA and Tukey’s post-hoc analysis (a=0.05). Results The addition of PG significantly increased setting time, flowability and Ca2+ release, but it compromised the hardness of the material. SEM showed that 50/50 group resulted porous material after setting due to the incomplete setting reaction, as shown by XRD analysis. The addition of PG (80/20 and 50/50) was not capable to improve cell proliferation or to enhance gene expression, and mineralized deposition of DPSC after 7 and 14 days as compared to the 100/0. Conclusion Except for flowability, the addition of PG did not promote further improvements on the chemical and physical properties evaluated, and it was not capable of enhancing the bioactivity of the MTA.

Pluripotency of Stem Cells from Human Exfoliated Deciduous Teeth for Tissue Engineering

Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative pluripotent cells that can be retrieved from primary teeth. Although SHED are isolated from the dental pulp, their differentiation potential is not limited to odontoblasts only. In fact, SHED can differentiate into several cell types including neurons, osteoblasts, adipocytes, and endothelial cells. The high plasticity makes SHED an interesting stem cell model for research in several biomedical areas. This review will discuss key findings about the characterization and differentiation of SHED into odontoblasts, neurons, and hormone secreting cells (e.g., hepatocytes and islet-like cell aggregates). The outcomes of the studies presented here support the multipotency of SHED and their potential to be used for tissue engineering-based therapies.

Graphene onto medical grade titanium: an atom-thick multimodal coating that promotes osteoblast maturation and inhibits biofilm formation from distinct species

Abstract The time needed for the osseointegration of titanium implants is deemed too long. Moreover, the bacterial colonization of their surfaces is a major cause of failure. Graphene can overcome these issues but its wet transfer onto substrates employs hazardous chemicals limiting the clinical applications. Alternatively, dry transfer technique has been developed, but the biological properties of this technique remain unexplored. Here, a dry transfer technique based on a hot-pressing method allowed to coat titanium substrates with high-quality graphene and coverage area >90% with a single transfer. The graphene-coated titanium is cytocompatible, did not induce cell membrane damage, induced human osteoblast maturation (gene and protein level), and increased the deposition of mineralized matrix compared to titanium alone. Moreover, graphene decreased the formation of biofilms from Streptococcus mutans, Enterococcus faecalis and even from whole saliva on titanium without killing the bacteria. These findings confirm that coating of titanium with graphene via a dry transfer technique is a promising strategy to improve osseointegration and prevent biofilm formation on implants and devices.

Graphene Nanosheets to Improve Physico-Mechanical Properties of Bioactive Calcium Silicate Cements

Bioactive calcium silicate cements are widely used to induce mineralization, to cement prosthetic parts, in the management of tooth perforations, and other areas. Nonetheless, they can present clinical disadvantages, such as long setting time and modest physico-mechanical properties. The objective of this work was to evaluate the potential of graphene nanosheets (GNS) to improve two bioactive cements. GNS were obtained via reduction of graphite oxide. GNS were mixed (1, 3, 5, and 7 wt %) with Biodentine (BIO) and Endocem Zr (ECZ), and the effects on setting time, hardness, push-out strength, pH profile, cell proliferation, and mineralization were evaluated. Statistics were performed with two-way ANOVA and Tukey test (α = 0.05). GNS has not interfered in the composition of the set cements as confirmed by Raman, FT-IR and XRD. GNS (1 and 3 wt %) shortened the setting time, increased hardness of both materials but decreased significantly the push-out strength of ECZ. pH was not affected but 1 wt % and 7 wt % to ECZ and 5 wt % to BIO increased the mineralization compared to the controls. In summary, GNS may be an alternative to improve the physico-mechanical properties and bioactivity of cements. Nonetheless, the use of GNS may not be advised for all materials when effective bonding is a concern.

Functional Odontoblastic-Like Cells Derived from Human iPSCs:

The induced pluripotent stem cells (iPSCs) have an intrinsic capability for indefinite self-renewal and large-scale expansion and can differentiate into all types of cells. Here, we tested the potential of iPSCs from dental pulp stem cells (DPSCs) to differentiate into functional odontoblasts. DPSCs were reprogrammed into iPSCs via electroporation of reprogramming factors OCT-4, SOX2, KLF4, LIN28, and L-MYC. The iPSCs presented overexpression of the reprogramming genes and high protein expressions of alkaline phosphatase, OCT4, and TRA-1-60 in vitro and generated tissues from 3 germ layers in vivo. Dentin discs with poly-L-lactic acid scaffolds containing iPSCs were implanted subcutaneously into immunodeficient mice. After 28 d from implantation, the iPSCs generated a pulp-like tissue with the presence of tubular dentin in vivo. The differentiation potential after long-term expansion was assessed in vitro. iPSCs and DPSCs of passages 4 and 14 were treated with either odontogenic medium or extract of bioactive cement for 28 d. Regardless of the passage tested, iPSCs expressed putative markers of odontoblastic differentiation and kept the same mineralization potential, while DPSC P14 failed to do the same. Analysis of these data collectively demonstrates that human iPSCs can be a source to derive human odontoblasts for dental pulp research and test bioactivity of materials.

PLGA nanoparticles as chlorhexidine-delivery carrier to resin-dentin adhesive interface

OBJECTIVE To characterize and deliver fabricated CHX-loaded PLGA-nanoparticles inside micron-sized dentinal-tubules of demineralized dentin-substrates and resin-dentin interface. METHODS Nanoparticles fabricated by emulsion evaporation were assessed in-vitro by different techniques. Delivery of drug-loaded nanoparticles to demineralized dentin substrates, interaction with collagen matrix, and ex-vivo CHX-release profiles using extracted teeth connected to experimental setup simulating pulpal hydrostatic pressure were investigated. Furthermore, nanoparticles association/interaction with a commercial dentin-adhesive applied to demineralized dentin substrates were examined. RESULTS The results showed that the formulated nanoparticles demonstrated attractive physicochemical properties, low cytotoxicity, potent antibacterial efficacy, and slow degradation and gradual CHX release profiles. Nanoparticles delivered efficiently inside dentinal-tubules structure to sufficient depth (>10μm) against the simulated upward pulpal hydrostatic-pressure, even after bonding-resins infiltration and were attached/retained on collagen-fibrils. These results verified the potential significance of this newly introduced drug-delivery therapeutic strategy for future clinical applications and promote for a new era of future dental research. SIGNIFICANCE This innovative drug-delivery strategy has proven to be a reliable method for delivering treatments that could be elaborated for other clinical applications in adhesive and restorative dentistry.

Graphene: An Emerging Carbon Nanomaterial for Bone Tissue Engineering

The development of materials and strategies that can promote faster bone healing and improved regeneration of bony defects is of high interest. Graphene and its derivatives (graphene oxide and reduced graphene oxide) have remarkable mechanical properties, can be chemically modified and allow the attachment of molecules and proteins. Due to these characteristics, these carbon-based materials have received increasing attention for several biomedical applications. As graphenes can improve mechanical properties of several biomaterials, induce, and increase cell differentiation toward osteoblasts, they have emerged as interesting alternatives for to promote bone regeneration. Herein, the key achievements made with graphenes for bone tissue engineering are presented with particular emphasis on their combination with biomaterials for bone regeneration and as coatings for biomedical implants.

CHAPTER 12:Smart Carbon Nanotubes and Graphenes for Tissue Engineering

Tissue engineering is an interdisciplinary field that merges the principles of engineering, biology and material sciences to develop approaches and therapeutic strategies to restore, replace or improve biological functions. The development of biomaterials plays a pivotal role in the progress of tissue engineering. Carbon nanotubes (CNTs) and graphene with its derivatives (graphene oxide and reduced graphene oxide) are carbon-based materials that present unique physical, chemical and mechanical properties. Besides having unparalleled electrical and mechanical properties, they can be modified via noncovalent or covalent attachment of molecules. Due to these distinct characteristics, these materials are interesting candidates to be used for various biomedical applications such as drug and gene delivery, biosensing, bioimaging and beyond. In tissue engineering, CNTs and graphenes can be used alone as substrates or mixed with other biomaterials to increase their bioactivity hence improving stem cell attachment, proliferation and differentiation towards various phenotypes. Herein, the advances made in CNTs and graphenes for tissue engineering are presented with particular emphasis on their biocompatibility and potential for tissue engineering and regeneration research.