Graphene Oxide Incorporated PLGA Nanofibrous Scaffold for Solid Phase Gene Delivery into Mesenchymal Stem Cells.

Delivery of functional genes into stem cells shows great application prospect in DNA-based tissue engineering. However, comparing with epithelial cells and cancer cells, stem cells usually exhibit low gene transfection efficiency. To enhance the transfection efficiency, non-viral gene delivery in combination with biomaterial scaffolds, has raised increasing interests from researchers in tissue engineering. Nanofibers fabricated by electrospinning technique mimicking extracellular matrix (ECM) are widely used in tissue engineering applications. In addition, graphene oxide (GO) with ultrahigh specific surface area and ultra-strong adsorption capability, is an ideal candidate for gene delivery. In this work, polyethylenimine (PEI)/plasmid DNA-GO/poly(D,L-lactic-co-glycolic acid) (PLGA) scaffold was developed as a substrate for solid phase gene delivery and a tissue engineering substrate for stem cells growth and differentiation. In order to improve the transfection efficiency of stem cells, PEI/pDNA complexes were immobilized at the surface of electropun GO incorporated PLGA nanofibrous mat. Human embryonic kidney 293 cells and human umbilical cord derived mesenchymal stem cells cultured on PEI/pDNA-GO/PLGA scaffold showed significantly higher green fluorescent protein (GFP) expression than PEI/pGFP in the medium. These findings demonstrated that solid phase gene delivery using PEI/pDNA-GO/PLGA significantly enhanced the gene transfection efficiency, and may find potential application of gene therapy and regeneration medicine.

Biodegradable Poly(aminoester)-Mediated p53 Gene Delivery for Cancer Therapy.

Gene therapy is a promising strategy in cancer treatment. However, efficient gene translation still remains challenging. In the previous work, a hydrolytically degradable poly(aminoester) with good biocompatibility was synthesized. Herein, the poly(aminoester) was explored as a vector for gene delivery and cancer therapy. The experiments revealed that the poly(aminoester) condensed plasmid DNA into nanosized particles via electrostatic interaction. The pEGFP-N1 and pGL-3 were first used as two reporter genes to study intracellular transfection. The poly(aminoester) showed higher GFP expression (33%) than PEI 25 kDa (21%). Intracellular trafficking of Cy3-labelled pGL-3 also indicated that the poly(aminoester) showed superior DNA delivery ability to nucleus compared to PEI 25 kDa. Furthermore, the therapeutic gene (p53) was translated into the breast cancer cell line (MCF-7), and then induced cell apoptosis. These results suggested that the degradable poly(aminoester) is a promising and efficient gene delivery vector for gene therapeutic applications.

A quality comparison of protein crystals grown under containerless conditions generated by diamagnetic levitation, silicone oil and agarose gel.

High-quality crystals are key to obtaining accurate three-dimensional structures of proteins using X-ray diffraction techniques. However, obtaining such protein crystals is often a challenge. Several containerless crystallization techniques have been reported to have the ability to improve crystal quality, but it is unknown which is the most favourable way to grow high-quality protein crystals. In this paper, a quality comparison of protein crystals which were grown under three containerless conditions provided by diamagnetic levitation, silicone oil and agarose gel was conducted. A control experiment on a vessel wall was also simultaneously carried out. Seven different proteins were crystallized under the four conditions, and the crystal quality was assessed in terms of the resolution limit, the mosaicity and the Rmerge. It was found that the crystals grown under the three containerless conditions demonstrated better morphology than those of the control. X-ray diffraction data indicated that the quality of the crystals grown under the three containerless conditions was better than that of the control. Of the three containerless crystallization techniques, the diamagnetic levitation technique exhibited the best performance in enhancing crystal quality. This paper is to our knowledge the first report of improvement of crystal quality using a diamagnetic levitation technique. Crystals obtained from agarose gel demonstrated the second best improvement in crystal quality. The study indicated that the diamagnetic levitation technique is indeed a favourable method for growing high-quality protein crystals, and its utilization is thus potentially useful in practical efforts to obtain well diffracting protein crystals.

Multifunctional hydrogel modulates the immune microenvironment to improve allogeneic spinal cord tissue survival for complete spinal cord injury repair.

Transplantation of allogeneic adult spinal cord tissues (aSCTs) to replace the injured spinal cord, serves as a promising strategy in complete spinal cord injury (SCI) repair. However, in addition to allograft immune rejection, damage-associated molecular pattern (DAMP)-mediated inflammatory microenvironments greatly impair the survival and function of transplants. In this study, we aimed to regulate the immune microenvironment after aSCT implantation by developing a functional hybrid gelatin and hyaluronic acid hydrogel (F-G/H) modified with cationic polymers and anti-inflammatory cytokines that can gelatinize at both ends of the aSCT to glue the grafts for perfect matching at defects. The F-G/H hydrogel exhibited the capacities of DAMP scavenging, sustainably released anti-inflammatory cytokines, and reduced lymphocyte accumulation, thereby modulating the immune response and enhancing the survival and function of aSCTs. When the hydrogel was used in combination with a systemic immunosuppressive drug treatment, the locomotor functions of SCI rats were significantly improved after aSCTs and F-G/H transplantation. This biomaterial-based immunomodulatory strategy may provide the potential for spinal cord graft replacement for treating SCI. STATEMENT OF SIGNIFICANCE: In this study, we aimed to regulate the immune microenvironment by developing a functional hybrid gelatin and hyaluronic acid hydrogel (F-G/H) modified with cationic polymers and anti-inflammatory cytokines that can gelatinize at both ends of the aSCT to glue the grafts for perfect matching at defects. We found that with the treatment of F-G/H hydrogel, the aSCT survival and function was significantly improved, as a result of reducing recruitment and activation of immune cells through TLR- and ST-2- related signaling. With the combination of immunosuppressive drug treatment, the locomotor functions of SCI rats were significantly improved after aSCTs and F-G/H transplantation. Findings from this work suggest the potential application of the F-G/H as a biomaterial-based immunoregulatory strategy for improving the therapeutic efficiency of the transplanted spinal cord graft for spinal cord injury repair.

Optimized, visible light-induced crosslinkable hybrid gelatin/hyaluronic acid scaffold promotes complete spinal cord injury repair.

Severe microenvironmental changes after spinal cord injury (SCI) present serious challenges in neural regeneration and tissue repair. Gelatin (GL)- and hyaluronic acid (HA)-based hydrogels are attractive scaffolds because they are major components of the extracellular matrix and can provide a favorable adjustable microenvironment for neurogenesis and motor function recovery. In this study, three-dimensional hybrid GL/HA hydrogel scaffolds were prepared and optimized. The hybrid hydrogels could undergoin situgelation and fit the defects perfectly via visible light-induced crosslinking in the complete SCI rats. We found that the transplantation of the hybrid hydrogel scaffold significantly reduced the inflammatory responses and suppressed glial scar formation in an HA concentration-dependent manner. Moreover, the hybrid hydrogel with GL/HA ratios less than 8/2 effectively promoted endogenous neural stem cell migration and neurogenesis, as well as improved neuron maturation and axonal regeneration. The results showed locomotor function improved 60 days after transplantation, thus suggesting that GL/HA hydrogels can be considered as a promising scaffold for complete SCI repair.

Acceleration of chondrogenic differentiation of human mesenchymal stem cells by sustained growth factor release in 3D graphene oxide incorporated hydrogels.

Damaged articular cartilage has limited self-healing capabilities, leading to degeneration that affects millions of people. Although cartilage tissue engineering is considered a promising approach for treatment, robust and long-term chondrogenesis within a 3-dimensional (3D) scaffold remains a major challenge for complete regeneration. Most current approaches involve incorporation of transforming growth factor-ß (TGF-ß) into the scaffold, but have limited utility owing to the short functional half-life and/or rapid clearance of TGF-ß. In this study, we have tested the incorporation of graphene oxide nanosheets (GO) within a photopolymerizable poly-D, l-lactic acid/polyethylene glycol (PDLLA) hydrogel, for its applicability in sustained release of the chondroinductive growth factor TGF-ß3. We found that with GO incorporation, the hydrogel scaffold (GO/PDLLA) exhibited enhanced initial mechanical strength, i.e., increased compressive modulus, and supported long-term, sustained release of TGF-ß3 for up to 4 weeks. In addition, human bone marrow-derived mesenchymal stem cells (hBMSCs) seeded within TGF-ß3 loaded GO/PDLLA hydrogels displayed high cell viability and improved chondrogenesis in a TGF-ß3 concentration-dependent manner. hBMSCs cultured in GO/PDLLA also demonstrated significantly higher chondrogenic gene expression, including aggrecan, collagen type II and SOX9, and cartilage matrix production when compared to cultures maintained in GO-free scaffolds containing equivalent amounts of TGF-ß3. Upon subcutaneous implantation in vivo, hBMSC-seeded TGF-ß3-GO/PDLLA hydrogel constructs displayed considerably greater cartilage matrix than their TGF-ß3/PDLLA counterparts without GO. Taken together, these findings support the potential application of GO in optimizing TGF-ß3 induced hBMSC chondrogenesis for cartilage tissue engineering. STATEMENT OF SIGNIFICANCE: In this work, we have developed a graphene oxide (GO) incorporated, photocrosslinked PDLLA hybrid hydrogel for localized delivery and sustained release of loaded TGF-ß3 to seeded cells. The incorporation of GO in PDLLA hydrogel suppressed the burst release of TGF-ß3, and significantly prolonged the retention time of the TGF-ß3 initially loaded in the hydrogel. Additionally, the GO improved the initial compressive strength of the hydrogel. Both in vitro analyses and in vivo implantation results showed that the GO/PDLLA constructs seeded with human mesenchymal stem cells (hMSCs) showed significantly higher cartilage formation, compared to GO-free scaffolds containing equivalent amount of TGF-ß3. Findings from this work suggest the potential application of the GO-TGF/PDLLA hydrogel as a functional scaffold for hMSC-based cartilage tissue engineering.

Recent developments in regenerative ophthalmology.

Regenerative medicine (RM) is one of the most promising disciplines for advancements in modern medicine, and regenerative ophthalmology (RO) is one of the most active fields of regenerative medicine. This review aims to provide an overview of regenerative ophthalmology, including the range of tools and materials being used, and to describe its application in ophthalmologic subspecialties, with the exception of surgical implantation of artificial tissues or organs (e.g., contact lens, artificial cornea, intraocular lens, artificial retina, and bionic eyes) due to space limitations. In addition, current challenges and limitations of regenerative ophthalmology are discussed and future directions are highlighted.

Accelerated biomineralization of graphene oxide - incorporated cellulose acetate nanofibrous scaffolds for mesenchymal stem cell osteogenesis.

For bone tissue engineering, it requires that the scaffolds have excellent biocompatibility, proper mechanical and osteoinductive properties. Electrospun nanofibers with extracellular matrices mimicking structure have proven to be good scaffolds for bone tissue repairing. Hybrid nanofibers in particular, endow the nanofibers with specific and multiple functionalities, and therefore have attracted increasing interests in the recent years. In this study, we fabricated graphene oxide (GO)-incorporated cellulose acetate (CA) nanofibrous scaffolds by electrospinning technique for enhancement of biomineralization and osteogenic differentiation of human mesenchymal stem cells (hMSCs). The results displayed the average fiber diameter was decreased from 595 to 285nm with the presence of GO from 0 to 1wt%. Furthermore, with incorporation of GO, the Young's modulus of the nanofibers increased in a dose-dependent manner. More importantly, the incorporation of GO led to significantly enhanced adhesion and proliferation of hMSCs on the scaffolds, mainly due to the good biocompatibility and extracellular matrices mimicking structure of the hybrid nanofibers. Exposure of the nanofibers to the simulated body fluid revealed that the biomineralization was improved significantly with the doping of GO in the nanofibers, possibly owing to the more nucleation sites for calcium phosphate provided by GO. The accelerated biomineralization on the GO-CA nanofibers resulted in a markedly increase in the activity of biomineralization-relevant alkaline phosphatase, and thus induced osteogenic differentiation of hMSCs. The current work demonstrated that the GO-CA nanofibrous scaffolds may find potential applications in bone tissue engineering and other regenerative medicine fields.

Chondrogenesis of human bone marrow mesenchymal stem cells in 3-dimensional, photocrosslinked hydrogel constructs: Effect of cell seeding density and material stiffness.

Three-dimensional hydrogel constructs incorporated with live stem cells that support chondrogenic differentiation and maintenance offer a promising regenerative route towards addressing the limited self-repair capabilities of articular cartilage. In particular, hydrogel scaffolds that augment chondrogenesis and recapitulate the native physical properties of cartilage, such as compressive strength, can potentially be applied in point-of-care procedures. We report here the synthesis of two new materials, [poly-l-lactic acid/polyethylene glycol/poly-l-lactic acid] (PLLA-PEG 1000) and [poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid] (PDLLA-PEG 1000), that are biodegradable, biocompatible (>80% viability post fabrication), and possess high, physiologically relevant mechanical strength (∼1500 to 1800kPa). This study examined the effects of physiologically relevant cell densities (4, 8, 20, and 50×106/mL) and hydrogel stiffnesses (∼150kPa to∼1500kPa Young's moduli) on chondrogenesis of human bone marrow stem cells incorporated in hydrogel constructs fabricated with these materials and a previously characterized PDLLA-PEG 4000. Results showed that 20×106cells/mL, under a static culture condition, was the most efficient cell seeding density for extracellular matrix (ECM) production on the basis of hydroxyproline and glycosaminoglycan content. Interestingly, material stiffness did not significantly affect chondrogenesis, but rather material concentration was correlated to chondrogenesis with increasing levels at lower concentrations based on ECM production, chondrogenic gene expression, and histological analysis. These findings establish optimal cell densities for chondrogenesis within three-dimensional cell-incorporated hydrogels, inform hydrogel material development for cartilage tissue engineering, and demonstrate the efficacy and potential utility of PDLLA-PEG 1000 for point-of-care treatment of cartilage defects. STATEMENT OF SIGNIFICANCE: Engineering cartilage with physiologically relevant mechanical properties for point-of-care applications represents a major challenge in orthopedics, given the generally low mechanical strengths of traditional hydrogels used in cartilage tissue engineering. In this study, we characterized a new material that possesses high mechanical strength similar to native cartilage, and determined the optimal cell density and scaffold stiffness to achieve the most efficient chondrogenic response from seeded human bone marrow stem cells. Results show robust chondrogenesis and strongly suggest the potential of this material to be applied clinically for point-of-care repair of cartilage defects.

Enhanced proliferation and osteogenic differentiation of mesenchymal stem cells on graphene oxide-incorporated electrospun poly(lactic-co-glycolic acid) nanofibrous mats.

Currently, combining biomaterial scaffolds with living stem cells for tissue regeneration is a main approach for tissue engineering. Mesenchymal stem cells (MSCs) are promising candidates for musculoskeletal tissue repair through differentiating into specific tissues, such as bone, muscle, and cartilage. Thus, successfully directing the fate of MSCs through factors and inducers would improve regeneration efficiency. Here, we report the fabrication of graphene oxide (GO)-doped poly(lactic-co-glycolic acid) (PLGA) nanofiber scaffolds via electrospinning technique for the enhancement of osteogenic differentiation of MSCs. GO-PLGA nanofibrous mats with three-dimensional porous structure and smooth surface can be readily produced via an electrospinning technique. GO plays two roles in the nanofibrous mats: first, it enhances the hydrophilic performance, and protein- and inducer-adsorption ability of the nanofibers. Second, the incorporated GO accelerates the human MSCs (hMSCs) adhesion and proliferation versus pure PLGA nanofiber and induces the osteogenic differentiation. The incorporating GO scaffold materials may find applications in tissue engineering and other fields.

The in vitro and in vivo toxicity of graphene quantum dots.

Graphene quantum dots (GQD) generate intrinsic fluorescence, and improves aqueous stability of graphene oxide (GO) while maintaining wide chemical adaptability and high adsorption capacity. Despite GO's remarkable advantages in bio-imaging, bio-sensing and other biomedical applications, its biosafety issues are still unclear. Here we report a detailed and systematic study on the in vitro and in vivo toxicity of GQD. The GQD sample was prepared through a facile oxidation approach and fully characterized by means of AFM, TEM, FTIR, XPS and elemental analysis. In vitro experiments showed that GQD exhibits very low cytotoxicity owing to its ultra-small size and high oxygen content. Then, the in vivo biodistribution experiment of GQD revealed no material accumulation in main organs of mice and fast clearance of GQD through kidney. In order to mimic clinic drug administration, mice were injected with GQD and GO (as comparison) multiple times for in vivo toxicity tests. We found that GQD showed no obvious influence on mice owing to its small size, while GO appeared toxic, even caused death to mice due to GO aggregation inside mice. In brief, GQD possesses no obvious in vitro and in vivo toxicity, even under multi-dosing situation.

Pilot study on efficacy of reduced cathartic bowel preparation with polyethylene glycol and bisacodyl.

AIM: To evaluate the efficacy of reduced cathartic bowel preparation with 2 L polyethylene glycol (PEG)-4000 electrolyte solution and 10 mg bisacodyl enteric-coated tablets for computed tomographic colonography (CTC). METHODS: Sixty subjects who gave informed consent were randomly assigned to study group A, study group B or the control group. On the day prior to CTC, subjects in study group A were given 20 mL 40% wt/vol barium sulfate suspension before 3 mealtimes, 60 mL 60% diatrizoate meglumine diluted in 250 mL water after supper, and 10 mg bisacodyl enteric-coated tablets 1 h before oral administration of 2 L PEG-4000 electrolyte solution. Subjects in study group B were treated identically to those in study group A, with the exception of bisacodyl which was given 1 h after oral PEG-4000. Subjects in the control group were managed using the same strategy as the subjects in study group A, but without administration of bisacodyl. Residual stool and fluid scores, the attenuation value of residual fluid, and discomfort during bowel preparation in the three groups were analyzed statistically. RESULTS: The mean scores for residual stool and fluid in study group A were lower than those in study group B, but the differences were not statistically significant. Subjects in study group A showed greater stool and fluid cleansing ability than the subjects in study group B. The mean scores for residual stool and fluid in study groups A and B were lower than those in the control group, and were significantly different. There was no significant difference in the mean attenuation value of residual fluid between study group A, study group B and the control group. The total discomfort index during bowel preparation was 46, 45 and 45 in the three groups, respectively, with no significant difference. CONCLUSION: Administration of 10 mg bisacodyl enteric-coated tablets prior to or after oral administration of 2 L PEG-4000 electrolyte solution enhances stool and fluid cleansing ability, and has no impact on the attenuation value of residual fluid or the discomfort index. The former is an excellent alternative for CTC colorectum cleansing.

PEGylated graphene oxide-mediated protein delivery for cell function regulation.

Delivery of proteins into cells may alter cellular functions as various proteins are involved in cellular signaling by activating or deactivating the corresponding pathways and, therefore, can be used in cancer therapy. In this study, we have demonstrated for the first time that PEGylated graphene oxide (GO) can be exploited as a nanovector for efficient delivery of proteins into cells. In this approach, GO was functionalized with amine-terminated 6-armed polyethylene glycol (PEG) molecules, thereby providing GO with proper physiological stability and biocompatibility. Proteins were then loaded onto PEG-grafted GO (GO-PEG) with high payload via noncovalent interactions. GO-PEG could deliver proteins to cytoplasm efficiently, protecting them from enzymatic hydrolysis. The protein delivered by GO-PEG reserves its biological activity that regulates the cell fate. As a result, delivery of ribonuclease A (RNase A) led to cell death and transport of protein kinase A (PKA) induced cell growth. Taken together, this work demonstrated the feasibility of PEGlyated GO as a promising protein delivery vector with high biocompatibility, high payload capacity and, more importantly, capabilities of protecting proteins from enzymatic hydrolysis and retaining their biological functions.