Graphene-Based Nanocomposites as Promising Options for Hard Tissue Regeneration.

Tissues are often damaged by physical trauma, infection or tumors. A slight injury heals naturally through the normal healing process, while severe injury causes serious health implications. Therefore, many efforts have been devoted to treat and repair various tissue defects. Recently, tissue engineering approaches have attracted a rapidly growing interest in biomedical fields to promote and enhance healing and regeneration of large-scale tissue defects. On the other hand, with the recent advances in nanoscience and nanotechnology, various nanomaterials have been suggested as novel biomaterials. Graphene, a two-dimensional atomic layer of graphite, and its derivatives have recently been found to possess promoting effects on various types of cells. In addition, their unique properties, such as outstanding mechanical and biological properties, allow them to be a promising option for hard tissue regeneration. Herein, we summarized recent research advances in graphene-based nanocomposites for hard tissue regeneration, and highlighted their promising potentials in biomedical and tissue engineering.

The Effect of Reduced Graphene Oxide-Coated Biphasic Calcium Phosphate Bone Graft Material on Osteogenesis.

This study was conducted to evaluate the effect of biphasic calcium phosphate (BCP) coated with reduced graphene oxide (rGO) as bone graft materials on bone regeneration. The rGO-coated BCP bone graft material was fabricatied by mixing rGO and BCP at various concentrations. The surface charge of rGO-coated BCP was measured to be -14.43 mV, which formed a static electrostatic interaction. Cell viabilities were significantly diminished at higher concentrations of ≥100 µg/mL. The calvarial defects of 48 rats were implanted rGO-coated BCPs at a weight ratio of 2:1000 (rGO2), 4:1000 (rGO4), and 10:1000 (rGO10), repectively. BCP was used as a control group. The micro-CT and histological analysis were performed to evaluate new bone formation at 2 and 8 weeks after surgery. The results showed that the new bone volume (mm³) was significantly higher in the experimental groups than in the control group. Histological analysis showed that new bone areas (%) were significantly higher in the rGO2 and rGO10 than in the control, and significantly higher in rGO4 than in the rGO2 and rGO10. Conclusively, the rGO-coated BCP was found to be effective on osteogenesis and the concentration of the composite was an important factor.

Stimulated myoblast differentiation on graphene oxide-impregnated PLGA-collagen hybrid fibre matrices.

BACKGROUND: Electrospinning is a simple and effective method for fabricating micro- and nanofiber matrices. Electrospun fibre matrices have numerous advantages for use as tissue engineering scaffolds, such as high surface area-to-volume ratio, mass production capability and structural similarity to the natural extracellular matrix (ECM). Therefore, electrospun matrices, which are composed of biocompatible polymers and various biomaterials, have been developed as biomimetic scaffolds for the tissue engineering applications. In particular, graphene oxide (GO) has recently been considered as a novel biomaterial for skeletal muscle regeneration because it can promote the growth and differentiation of myoblasts. Therefore, the aim of the present study was to fabricate the hybrid fibre matrices that stimulate myoblasts differentiation for skeletal muscle regeneration. RESULTS: Hybrid fibre matrices composed of poly(lactic-co-glycolic acid, PLGA) and collagen (Col) impregnated with GO (GO-PLGA-Col) were successfully fabricated using an electrospinning process. Our results indicated that the GO-PLGA-Col hybrid matrices were comprised of randomly-oriented continuous fibres with a three-dimensional non-woven porous structure. Compositional analysis showed that GO was dispersed uniformly throughout the GO-PLGA-Col matrices. In addition, the hydrophilicity of the fabricated matrices was significantly increased by blending with a small amount of Col and GO. The attachment and proliferation of the C2C12 skeletal myoblasts were significantly enhanced on the GO-PLGA-Col hybrid matrices. Furthermore, the GO-PLGA-Col matrices stimulated the myogenic differentiation of C2C12 skeletal myoblasts, which was enhanced further under the culture conditions of the differentiation media. CONCLUSIONS: Taking our findings into consideration, it is suggested that the GO-PLGA-Col hybrid fibre matrices can be exploited as potential biomimetic scaffolds for skeletal tissue engineering and regeneration because these GO-impregnated hybrid matrices have potent effects on the induction of spontaneous myogenesis and exhibit superior bioactivity and biocompatibility.

Hyaluronic acid/poly(lactic-co-glycolic acid) core/shell fiber meshes loaded with epigallocatechin-3-O-gallate as skin tissue engineering scaffolds.

In this study, hyaluronic acid (HA)/poly(lactic-co-glycolic acid, PLGA) core/shell fiber meshes loaded with epigallocatechin-3-O-gallate (EGCG) (HA/PLGA-E) for application to tissue engineering scaffolds for skin regeneration were prepared via coaxial electrospinning. Physicochemical properties of HA/PLGA-E core/shell fiber meshes were characterized by SEM, Raman spectroscopy, contact angle, EGCG release profiling and in vitro degradation. Biomechanical properties of HA/PLGA-E meshes were also investigated by a tensile strength test. SEM images showed that HA/PLGA-E fiber meshes had a three-dimensional interconnected pore structure with an average fiber diameter of about 1270 nm. Raman spectra revealed that EGCG was uniformly dispersed in the PLGA shell of meshes. HA/PLGA-E meshes showed sustained EGCG release patterns by controlled diffusion and PLGA degradation over 4 weeks. EGCG loading did not adversely affect the tensile strength and elastic modulus of HA/PLGA meshes, while increased their hydrophilicity and surface energy. Attachment of human dermal fibroblasts on HA/PLGA-E meshes was appreciably increased and their proliferation was steadily retained during the culture period. These results suggest that HA/PLGA-E core/shell fiber meshes can be potentially used as scaffolds supporting skin regeneration.

Membrane reciprocation and quorum quenching: An innovative combination for fouling control and energy saving in membrane bioreactors.

Membrane bioreactors (MBRs) play a crucial role in wastewater treatment, but they face considerable challenges due to fouling. To tackle this issue, innovative strategies are needed. This study investigated the effectiveness of membrane reciprocation and quorum quenching (QQ) to control fouling in MBRs. The study compared MBRs using membrane reciprocation (30 rpm) and QQ (injecting media containing 100 or 200 mg/L BH4) with conventional MBRs employing different air-scouring intensities. The results demonstrated that combining membrane reciprocation (30 rpm) with QQ (200 mg/L BH4) significantly extended the service time of MBRs, making it approximately six times longer than conventional methods. Moreover, this approach reduced physically reversible resistance. The reduction in signal molecules related to biofouling due to QQ showcased its critical role in controlling biofouling, even under high shear caused by membrane reciprocation. However, the impact of QQ on microbial community structure appeared relatively insignificant when compared to factors such as operation time, aeration intensity, and membrane reciprocation. By combining membrane reciprocation and QQ, the study achieved a remarkable 81 % energy saving compared to extensive aeration (103 s-1 in velocity gradient), in addition to the extended service time. Importantly, this combined antifouling approach did not negatively affect microbial characteristics and wastewater treatment, emphasizing its effectiveness in MBRs. Overall, the findings of this study offer valuable insights for developing synergistic fouling control strategies in MBRs, significantly improving the energy efficiency of the wastewater treatment process.

Cell imaging and DNA delivery in fibroblastic cells by conjugated polyelectrolytes.

This study concentrates on the potential application of conjugated polyelectrolytes (CPEs) to cell imaging and DNA delivery. Four different types of polyfluorene copolymers, namely, PAHFP-Br, PAEFP-Br, PAHFbT-Br, and PSBFP-Na, which have the same π-conjugated backbone but different side chains, were synthesized. For cytotoxicity testing, L-929 fibroblastic cells were treated with increasing concentrations (0-50 µM) of each CPE and then cell viability was determined by WST-8 assay. Cellular uptake of CPEs into cultured L-929 cells was observed by fluorescence microscopy. To examine DNA delivery by CPEs, the cells were incubated for 1 H with PAHFP-Br/fluorescein (Fl)-labeled single-stranded DNA (ssDNA-Fl) complex and then visualized by fluorescence microscopy. Cytotoxicity of CPEs was increased in a dose-dependent manner but at lower than 10 µM, PAHFP-Br, PAEFP-Br, and PSBFP-Na did not show any cytotoxic effects on the cells. When added to cell cultures at 1 µM, PAHFP-Br/ssDNA-Fl complex was delivered and then dissociated into PAHFP-Br and ssDNA-Fl within the cells. This result implies that PAHFP-Br can enable cell imaging and DNA delivery into fibroblastic cells. Therefore, it is suggested that PAHFP-Br with various advantages such as low cytotoxicity and high fluorescence efficiency can be extensively used as a potential agent for cell imaging and gene delivery.

Enhanced osseointegration of dental implants with reduced graphene oxide coating.

BACKGROUND: The implants of pure titanium (Ti) and its alloys can lead to implant failure because of their poor interaction with bone-associated cells during bone regeneration. Surface modification over implants has achieved successful implants for enhanced osseointegration. Herein, we report a robust strategy to implement bioactive surface modification for implant interface enabled by the combinatorial system of reduced graphene oxide (rGO)-coated sandblasted, large-grit, and acid-etched (SLA) Ti to impart benefits to the implant. METHODS: We prepared SLA Ti (ST) implants with different surface modifications [i.e., rGO and recombinant human bone morphogenetic protein-2 (rhBMP-2)] and investigated their dental tissue regenerating ability in animal models. We performed comparative studies in surface property, in vitro cellular behaviors, and in vivo osseointegration activity among different groups, including ST (control), rhBMP-2-immobilized ST (BI-ST), rhBMP-2-treated ST (BT-ST), and rGO-coated ST (R-ST). RESULTS: Spectroscopic, diffractometric, and microscopic analyses confirmed that rGO was coated well around the surfaces of Ti discs (for cell study) and implant fixtures (for animal study). Furthermore, in vitro and in vivo studies revealed that the R-ST group showed significantly better effects in cell attachment and proliferation, alkaline phosphatase activity, matrix mineralization, expression of osteogenesis-related genes and protein, and osseointegration than the control (ST), BI-ST, and BT-ST groups. CONCLUSION: Hence, we suggest that the rGO-coated Ti can be a promising candidate for the application to dental or even orthopedic implants due to its ability to accelerate the healing rate with the high potential of osseointegration.

Nasolacrimal stent with shape memory as an advanced alternative to silicone products.

Epiphora is the overflow of tears typically caused by obstruction or occlusion of the nasolacrimal duct. More attention is required to address this global health issue owing to the increase in air pollution. Implantation of a silicone stent is the preferred treatment for epiphora; however, introducing a silicone stent into a narrow duct with complex geometry is challenging as it requires guidance by a sharp metal needle. Additionally, silicone can cause adverse reactions such as biofilm formation and tear flow resistance due to its extreme hydrophobicity. To overcome these problems, in this study we developed a new type of biocompatible shape memory polymer (SMP) stent with elasticity capacity for self-expansion. First, SMPs in the form of x%poly(ε-caprolactone)-co-y%poly(glycidyl methacrylate) (x%PCL-y%PGMA) were synthesized via ring opening polymerization by varying the molar ratio of PCL (x%) and PGMA (y%). Second, the shape memory and mechanical properties were tuned by controlling the crosslinking degree and concentration of x%PCL-y%PGMA solution to produce a test type of SMP stent. Lastly, this 94%PCL-06%PGMA stent exhibited more standout critical functions in a series of in vitro and in vivo experiments such as a cell growth-supporting level of biocompatibility with nasal epithelial cells without significant inflammatory responses, better resistance to biofilm formation, and more efficient capacity to drain tear than the silicone control. Overall, 94%PCL-06%PGMA can be suggested as a superior alternative to the currently used materials for nasolacrimal stents. STATEMENT OF SIGNIFICANCE: Silicone intubation (stenting) has been widely used to treat nasolacrimal duct obstruction, however, it can cause adverse clinical effects such as bacterial infection; presents procedural challenges because of the curved nasolacrimal duct structure; and shows poor drainage efficiency stemming from the highly hydrophobic nature of silicone. In this work, we describe an innovative shape memory polymer (SMP) as a superior alternative to conventional silicone-based materials for nasolacrimal duct intubation. We demonstrate the clear advantages of the SMP over conventional silicone, including a much higher drainage capacity and superior resistance to bacterial infection.

Enhanced osteogenic differentiation of human mesenchymal stem cells on Ti surfaces with electrochemical nanopattern formation.

Titanium (Ti) and its alloys are mainly used for dental and orthopedic applications due to their excellent biocompatibility and mechanical properties. However, their intrinsic bioinertness often quotes as a common complaint for biomedical applications. Herein, we produced nanopattern Ti surfaces with 10 nm nanopores in 120 nm dimples by electrochemical nanopattern formation (ENF), and evaluated the osteogenic differentiation of human mesenchymal stem cells (hMSCs) on the nanopattern Ti surfaces. The ENF surfaces were obtained by removing the TiO2 nanotube (NT) layers prepared by an anodization process. To determine the in vitro effects of the ENF surface, cell proliferation assay, alkaline phosphatase activity assay, alizarin red staining, western blotting, and immunocytochemistry were performed. Atomic force microscopy and scanning electron microscopy analysis show that the ENF surface has an ultrafine surface roughness with highly aligned nanoporous morphology. hMSCs on ENF surfaces exhibit increased proliferation and enhanced osteogenic differentiation as compared to the ordered TiO2 nanotubular and compact TiO2 surfaces. Surface modification with the ENF process is a promising technique for fabricating osteointegrative implant materials with a highly bioactive, rigid and purified nano surfaces.

Development of a Shape-Memory Tube to Prevent Vascular Stenosis.

Inserting a graft into vessels with different diameters frequently causes severe damage to the host vessels. Poor flow patency is an unresolved issue in grafts, particularly those with diameters less than 6 mm, because of vessel occlusion caused by disturbed blood flow following fast clotting. Herein, successful patency in the deployment of an ≈2 mm diameter graft into a porcine vessel is reported. A new library of property-tunable shape-memory polymers that prevent vessel damage by expanding the graft diameter circumferentially upon implantation is presented. The polymers undergo seven consecutive cycles of strain energy-preserved shape programming. Moreover, the new graft tube, which features a diffuser shape, minimizes disturbed flow formation and prevents thrombosis because its surface is coated with nitric-oxide-releasing peptides. Improved patency in a porcine vessel for 18 d is demonstrated while occlusive vascular remodeling occurs. These insights will help advance vascular graft design.

Three-dimensional graphene oxide-coated polyurethane foams beneficial to myogenesis.

The development of three dimensional (3D) scaffolds for promoting and stimulating cell growth is one of the greatest concerns in biomedical and tissue engineering. In the present study, novel biomimetic 3D scaffolds composed of polyurethane (PU) foam and graphene oxide (GO) nanosheets were designed, and their potential as 3D scaffolds for skeletal tissue regeneration was explored. The GO-coated PU foams (GO-PU foams) were characterized by scanning electron microscopy and Raman spectroscopy. It was revealed that the 3D GO-PU foams consisted of an interconnected foam-like network structure with an approximate 300 µm pore size, and the GO was uniformly distributed in the PU foams. On the other hand, the myogenic stimulatory effects of GO on skeletal myoblasts were also investigated. Moreover, the cellular behaviors of the skeletal myoblasts within the 3D GO-PU foams were evaluated by immunofluorescence analysis. Our findings showed that GO can significantly promote spontaneous myogenic differentiation without any myogenic factors, and the 3D GO-PU foams can provide a suitable 3D microenvironment for cell growth. Furthermore, the 3D GO-PU foams stimulated spontaneous myogenic differentiation via the myogenic stimulatory effects of GO. Therefore, this study suggests that the 3D GO-PU foams are beneficial to myogenesis, and can be used as biomimetic 3D scaffolds for skeletal tissue engineering.

Aligned laminin core-polydioxanone/collagen shell fiber matrices effective for neuritogenesis.

Neural tissue regeneration is a significant challenge, because severe nerve injury is quite difficult to regenerate spontaneously. Although, many studies have been devoted to promote nerve regeneration, there are still many technical challenges to achieve satisfactory results. In this study, we designed biomimetic matrices composed of aligned laminin core-polydioxanone/collagen shell (Lam-PDO/Col) fibers, which can provide both topographical and biochemical cues for promoting neuritogenesis. The aligned Lam-PDO/Col core-shell fiber matrices were fabricated by magnetic field-assisted electrospinning with the coaxial system, and their potential as biofunctional scaffolds for promoting neuritogenesis was explored. It was demonstrated that the aligned Lam-PDO/Col core-shell fibers were successfully fabricated, and the laminin in the core of fibers was steadily and continuously released from fibers. In addition, the cellular behaviors of hippocampal neuronal cells on the matrices were significantly enhanced. Moreover, the aligned Lam-PDO/Col fiber matrices effectively improved and guided neurite outgrowth as well as the neurogenic differentiation by providing both topographical and biochemical cues through aligned fiber structure and sustained release of laminin. Collectively, it is suggested that the aligned Lam-PDO/Col core-shell fiber matrices are one of the most promising approaches for promoting neuritogenesis and neural tissue regeneration.

Hyaluronic Acid/PLGA Core/Shell Fiber Matrices Loaded with EGCG Beneficial to Diabetic Wound Healing.

During the last few decades, considerable research on diabetic wound healing strategies has been performed, but complete diabetic wound healing remains an unsolved problem, which constitutes an enormous biomedical burden. Herein, hyaluronic acid (HA)/poly(lactic-co-glycolic acid, PLGA) core/shell fiber matrices loaded with epigallocatechin-3-O-gallate (EGCG) (HA/PLGA-E) are fabricated by coaxial electrospinning. HA/PLGA-E core/shell fiber matrices are composed of randomly-oriented sub-micrometer fibers and have a 3D porous network structure. EGCG is uniformly dispersed in the shell and sustainedly released from the matrices in a stepwise manner by controlled diffusion and PLGA degradation over four weeks. EGCG does not adversely affect the thermomechanical properties of HA/PLGA-E matrices. The number of human dermal fibroblasts attached on HA/PLGA-E matrices is appreciably higher than that on HA/PLGA counterparts, while their proliferation is steadily retained on HA/PLGA-E matrices. The wound healing activity of HA/PLGA-E matrices is evaluated in streptozotocin-induced diabetic rats. After two weeks of surgical treatment, the wound areas are significantly reduced by the coverage with HA/PLGA-E matrices resulting from enhanced re-epithelialization/neovascularization and increased collagen deposition, compared with no treatment or HA/PLGA. In conclusion, the HA/PLGA-E matrices can be potentially exploited to craft strategies for the acceleration of diabetic wound healing and skin regeneration.

Reduced graphene oxide-coated hydroxyapatite composites stimulate spontaneous osteogenic differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have great potential as cell sources for bone tissue engineering and regeneration, but the control and induction of their specific differentiation into bone cells remain challenging. Graphene-based nanomaterials are considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for SC differentiation and components of implantable devices, due to their biocompatible and bioactive properties. Despite the potential biomedical applications of graphene and its derivatives, only limited information is available regarding their osteogenic activity. This study concentrates upon the effects of reduced graphene oxide (rGO)-coated hydroxyapatite (HAp) composites on osteogenic differentiation of hMSCs. The average particle sizes of HAp and rGO were 1270 ± 476 nm and 438 ± 180 nm, respectively. When coated on HAp particulates, rGO synergistically enhanced spontaneous osteogenic differentiation of hMSCs, without hampering their proliferation. This result was confirmed by determining alkaline phosphatase activity and mineralization of calcium and phosphate as early and late stage markers of osteogenic differentiation. It is suggested that rGO-coated HAp composites can be effectively utilized as dental and orthopedic bone fillers since these graphene-based particulate materials have potent effects on stimulating the spontaneous differentiation of MSCs and show superior bioactivity and osteoinductive potential.

Epigallocatechin-3-O-Gallate-Loaded Poly(lactic-co-glycolic acid) Fibrous Sheets as Anti-Adhesion Barriers.

Epigallocatechin-3-O-gallate (EGCG), the main polyphenolic component of green tea, has a wide range of pharmacological activities, including antioxidant, anti-inflammatory, and anti-fibrotic effects. In this study, EGCG-loaded poly(lactic-co-glycolic acid) (PLGA) sheets were prepared by electrospinning nanofibers and evaluating their potential as tissue-adhesion barriers. EGCG-loaded PLGA (E-PLGA) fibrous sheets were electrospun from a PLGA solution containing 8% (w/v) EGCG. The average diameter of E-PLGA fibers was 397 ± 159 nm, which was comparable to that of pure PLGA fibers (459 ± 154 nm). EGCG was uniformly dispersed in E-PLGA sheets without direct chemical interactions. E-PLGA fibrous sheets showed sustained release of EGCG by controlled diffusion and PLGA degradation. The attachment and proliferation of L-929 fibroblastic cells were significantly (p < 0.05) suppressed in E-PLGA sheets. Furthermore, E-PLGA fibrous sheets did not induce any inflammatory response to J774A.1 macrophages. The anti-adhesion efficacy of E-PLGA fibrous sheets was evaluated in the intraperitoneal adhesion model in rats. Two weeks after surgical treatment, macroscopic adhesion (extent and severity) scores and histopathological tissue responses of E-PLGA fibrous sheets were significantly lower than those of non-treated controls and pure PLGA sheets. The results suggest that the scores are comparable, and in some cases superior, to those of other commercialized tissue-adhesion barriers. In conclusion, our study findings suggest that E-PLGA fibrous sheets may be exploited as potential tissue-adhesion barriers for the prevention of post-surgical adhesion formation.

Enhanced Osteogenesis by Reduced Graphene Oxide/Hydroxyapatite Nanocomposites.

Recently, graphene-based nanomaterials, in the form of two dimensional substrates or three dimensional foams, have attracted considerable attention as bioactive scaffolds to promote the differentiation of various stem cells towards specific lineages. On the other hand, the potential advantages of using graphene-based hybrid composites directly as factors inducing cellular differentiation as well as tissue regeneration are unclear. This study examined whether nanocomposites of reduced graphene oxide (rGO) and hydroxyapatite (HAp) (rGO/HAp NCs) could enhance the osteogenesis of MC3T3-E1 preosteoblasts and promote new bone formation. When combined with HAp, rGO synergistically promoted the spontaneous osteodifferentiation of MC3T3-E1 cells without hindering their proliferation. This enhanced osteogenesis was corroborated from determination of alkaline phosphatase activity as early stage markers of osteodifferentiation and mineralization of calcium and phosphate as late stage markers. Immunoblot analysis showed that rGO/HAp NCs increase the expression levels of osteopontin and osteocalcin significantly. Furthermore, rGO/HAp grafts were found to significantly enhance new bone formation in full-thickness calvarial defects without inflammatory responses. These results suggest that rGO/HAp NCs can be exploited to craft a range of strategies for the development of novel dental and orthopedic bone grafts to accelerate bone regeneration because these graphene-based composite materials have potentials to stimulate osteogenesis.

PLGA nanofiber membranes loaded with epigallocatechin-3-O-gallate are beneficial to prevention of postsurgical adhesions.

This study concentrates on the development of biodegradable nanofiber membranes with controlled drug release to ensure reduced tissue adhesion and accelerated healing. Nanofibers of poly(lactic-co-glycolic acid) (PLGA) loaded with epigallocatechin-3-O-gallate (EGCG), the most bioactive polyphenolic compound in green tea, were electrospun. The physicochemical and biomechanical properties of EGCG-releasing PLGA (E-PLGA) nanofiber membranes were characterized by atomic force microscopy, EGCG release and degradation profiles, and tensile testing. In vitro antioxidant activity and hemocompatibility were evaluated by measuring scavenged reactive oxygen species levels and activated partial thromboplastin time, respectively. In vivo antiadhesion efficacy was examined on the rat peritonea with a surgical incision. The average fiber diameter of E-PLGA membranes was approximately 300-500 nm, which was almost similar to that of pure PLGA equivalents. E-PLGA membranes showed sustained EGCG release mediated by controlled diffusion and PLGA degradation over 28 days. EGCG did not adversely affect the tensile strength of PLGA membranes, whereas it significantly decreased the elastic modulus and increased the strain at break. E-PLGA membranes were significantly effective in both scavenging reactive oxygen species and extending activated partial thromboplastin time. Macroscopic observation after 1 week of surgical treatment revealed that the antiadhesion efficacy of E-PLGA nanofiber membranes was significantly superior to those of untreated controls and pure PLGA equivalents, which was comparable to that of a commercial tissue-adhesion barrier. In conclusion, the E-PLGA hybrid nanofiber can be exploited to craft strategies for the prevention of postsurgical adhesions.