Fabrication and Evaluation of Gellan Gum/Hyaluronic Acid Hydrogel for Retinal Tissue Engineering Biomaterial and the Influence of Substrate Stress Relaxation on Retinal Pigment Epithelial Cells.

Cell therapies for age-related macular degeneration (AMD) treatment have been developed by integrating hydrogel-based biomaterials. Until now, cell activity has been observed only in terms of the modulus of the hydrogel. In addition, cell behavior has only been observed in the 2D environment of the hydrogel and the 3D matrix. As time-dependent stress relaxation is considered a significant mechanical cue for the control of cellular activities, it is important to optimize hydrogels for retinal tissue engineering (TE) by applying this viewpoint. Herein, a gellan Gum (GG)/Hyaluronic acid (HA) hydrogel was fabricated using a facile physical crosslinking method. The physicochemical and mechanical properties were controlled by forming a different composition of GG and HA. The characterization was performed by conducting a mass swelling study, a sol fraction study, a weight loss test, a viscosity test, an injection force study, a compression test, and a stress relaxation analysis. The biological activity of the cells encapsulated in 3D constructs was evaluated by conducting a morphological study, a proliferation test, a live/dead analysis, histology, immunofluorescence staining, and a gene expression study to determine the most appropriate material for retinal TE biomaterial. Hydrogels with moderate amounts of HA showed improved physicochemical and mechanical properties suitable for injection into the retina. Moreover, the time-dependent stress relaxation property of the GG/HA hydrogel was enhanced when the appropriate amount of HA was loaded. In addition, the cellular compatibility of the GG/HA hydrogel in in vitro experiments was significantly improved in the fast-relaxing hydrogel. Overall, these results demonstrate the remarkable potential of GG/HA hydrogel as an injectable hydrogel for retinal TE and the importance of the stress relaxation property when designing retinal TE hydrogels. Therefore, we believe that GG/HA hydrogel is a prospective candidate for retinal TE biomaterial.

Preparation and evaluation of gellan gum hydrogel reinforced with silk fibers with enhanced mechanical and biological properties for cartilage tissue engineering.

Various research about cartilage regeneration using biomaterials has been done recently. Particularly, gellan gum hydrogel (GG) is reported to be suitable as a biomaterial for cartilage tissue engineering (TE) for its water uptaking ability, producibility, and environmental resemblance of native cartilage. Despite these advantages, mechanical and cell adhesion properties are still difficult to modulate. Reinforcement is essential to overcome these problems. Herein, GG was modified by physically blending with different lengths of silk fiber (SF). As SF is expected to improve such disadvantages of GG, mechanical and biological properties were characterized to confirm its reinforcement ability. Mechanical properties such as degradation rate, swelling rate, compression strength, and viscosity were studied and it was confirmed that SF significantly reinforces the mechanical properties of GG. Furthermore, in vitro study was carried out to confirm morphology, biocompatibility, proliferation, and chondrogenesis of chondrocytes encapsulated in the hydrogels. Overall, chondrocytes in the GG blended with SF (SF/GG) showed enhanced cell viability and growth. According to this study, SF/GG can be a promising biomaterial for cartilage TE biomaterial.

Development and Evaluation of Gellan Gum/Silk Fibroin/Chondroitin Sulfate Ternary Injectable Hydrogel for Cartilage Tissue Engineering.

Hydrogel is in the spotlight as a useful biomaterial in the field of drug delivery and tissue engineering due to its similar biological properties to a native extracellular matrix (ECM). Herein, we proposed a ternary hydrogel of gellan gum (GG), silk fibroin (SF), and chondroitin sulfate (CS) as a biomaterial for cartilage tissue engineering. The hydrogels were fabricated with a facile combination of the physical and chemical crosslinking method. The purpose of this study was to find the proper content of SF and GG for the ternary matrix and confirm the applicability of the hydrogel in vitro and in vivo. The chemical and mechanical properties were measured to confirm the suitability of the hydrogel for cartilage tissue engineering. The biocompatibility of the hydrogels was investigated by analyzing the cell morphology, adhesion, proliferation, migration, and growth of articular chondrocytes-laden hydrogels. The results showed that the higher proportion of GG enhanced the mechanical properties of the hydrogel but the groups with over 0.75% of GG exhibited gelling temperatures over 40 °C, which was a harsh condition for cell encapsulation. The 0.3% GG/3.7% SF/CS and 0.5% GG/3.5% SF/CS hydrogels were chosen for the in vitro study. The cells that were encapsulated in the hydrogels did not show any abnormalities and exhibited low cytotoxicity. The biochemical properties and gene expression of the encapsulated cells exhibited positive cell growth and expression of cartilage-specific ECM and genes in the 0.5% GG/3.5% SF/CS hydrogel. Overall, the study of the GG/SF/CS ternary hydrogel with an appropriate content showed that the combination of GG, SF, and CS can synergistically promote articular cartilage defect repair and has considerable potential for application as a biomaterial in cartilage tissue engineering.

Pluronic F-127/Silk Fibroin for Enhanced Mechanical Property and Sustained Release Drug for Tissue Engineering Biomaterial.

Herein, an injectable thermosensitive hydrogel was developed for a drug and cellular delivery system. The composite was prepared by facile physical mixing of pluronic F-127 (PF) and silk fibroin (SF) in an aqueous solution. The chemical structure, transparency, viscosity, injectability, degradation kinetic, cumulative release of dexamethasone (Dex), a type of corticosteroid drug, and size distribution of the fabricated hydrogels were characterized. Cytotoxicity of the hydrogels was also studied to verify the biocompatibility of the hydrogels. The addition of a proper amount of SF to PF not only improved the mechanical strength but also decreased the degradation rate which improved the fast rate release of hydrophobic drugs. The cytotoxicity of the hydrogel decreased when SF was added to PF in a proper amount. Overall, the results confirm that the composite of PF and SF can be a promising cell and drug delivery system for future application in tissue engineering and regenerative medicine.

Dopamine-Functionalized Gellan Gum Hydrogel as a Candidate Biomaterial for a Retinal Pigment Epithelium Cell Delivery System.

In this study, dopamine-functionalized gellan gum (DFG) hydrogel was prepared as a carrier for retinal pigment epithelium (RPE) cell delivery via a carbodiimide reaction. The carboxylic acid of gellan gum (GG) was replaced with catechol in a 21.3% yield, which was confirmed by NMR. Sol fraction and weight loss measurements revealed that dopamine improved degradability in the GG hydrogel. Measurements of the viscosity, injection force, and compressibility also showed that dopamine-functionalized GG hydrogels had more desirable rheological/mechanical properties for improving injectability. These characteristics were confirmed to arise from the GG's helix structure loosened by the dopamine's bulky nature. Moreover, dopamine's hydrophilic characteristics were confirmed to create a more favorable microenvironment for cell growth by promoting swelling capability and cell attachment. This improved biocompatibility became more pronounced when the hydrophilicity of dopamine was combined with a larger specific surface area stemming from the less porous structure of the dopamine-grafted hydrogels. This effect was apparent from the live/dead staining images of the as-prepared hydrogels. Meanwhile, the nonionic cross-linked DFG (DG) hydrogel showed the lowest protein expression in the immunofluorescence staining images obtained after 28 days of culture, supporting that it had the highest degradability and associated cell-releasing ability. That tendency was also observed in the gene expression data acquired by real-time polymerase chain reaction (RT-PCR) analysis. RT-PCR analysis also revealed that the DG hydrogel carrier could upregulate the visual function-related gene of RPE. Overall, the DG hydrogel system demonstrated its feasibility as a carrier of RPE cells and its potential as a means of improving visual function.

Preparation and characterization of an injectable dexamethasone-cyclodextrin complexes-loaded gellan gum hydrogel for cartilage tissue engineering.

In this study, 6-(6-aminohexyl) amino-6-deoxy-ß-cyclodextrin-gellan gum complex hydrogel (HCD-GG) was developed to enhance the affinity of anti-inflammatory drug dexamethasone (Dx), improve chondrogenesis, and decrease the inflammatory response. The modified chemical structure was confirmed by NMR and FTIR. Mechanical and physicochemical properties were characterized by performing viscosity study, compression test, injection force test, swelling kinetic, weight loss, and morphological study. The release profile of the drug-loaded hydrogels was analyzed to confirm the affinity of the hydrophobic drugs and the matrix and characterize cumulative release. In vitro test was carried out with MTT assay, live/dead staining, glycosaminoglycan (GAGs) content, double-stranded DNA (dsDNA) content, morphological analysis, histology, and gene expression. In vivo experiment was conducted by implanting the samples under a subcutaneous area of SPD rat and cartilage defected rabbit model. The results displayed successfully synthesized HCD-GG. The gelation temperature of the modified hydrogels was decreased while the mechanical property was improved when the drug was loaded in the modified hydrogel. Swelling and degradation kinetics resulted in a higher level compared to the pristine GG but was a sufficient level to support drugs and cells. The affinity and release rate of the drug was higher in the HCD-GG group which shows an improved drug delivery system of the GG-based material. The microenvironment provided a suitable environment for cells to grow. Also, chondrogenesis was affected by the existence of Dx and microenvironment, resulting in higher expression levels of cartilage-related genes while the expression of the inflammation mediators decreased when the Dx was loaded. In vivo study showed an improved anti-inflammatory response in the drug-loaded hydrogel. Furthermore, the cartilage defected rabbit model showed an enhanced regenerative effect when the Dx@HCD-GG was implanted. These results suggest that HCD-GG and Dx@HCD-GG have the potential for cartilage regeneration along with multiple applications in tissue engineering and regenerative medicine.

Advanced gellan gum-based glycol chitosan hydrogel for cartilage tissue engineering biomaterial.

Gellan gum (GG), a nature-derived polysaccharide, is one of the materials widely used in cartilage tissue engineering (TE). Glycol chitosan (GC), a derivative of chitosan, is a water-soluble natural polymer that has excellent biocompatibility and biodegradability as well as cell adhesion. Herein, GG was physically blended with GC to enhance the mechanical properties and microenvironment of the GG to apply in cartilage TE. The study was conducted with a hydrogel model which is similar to the extracellular matrix (ECM) of cartilage tissue. The physicochemical studies were carried out with morphological study, swelling ratio, weight loss, and sol fraction. The mechanical characterization was conducted with compression test and rheological study to confirm availability in cartilage TE material. Furthermore, in vitro studies such as morphology investigation, viability assay, GAG content, qRT-PCR, and histological study were performed to verify biocompatibility and chondrogenesis of the material. The mechanical and biological properties improved with a proper amount of GC. Overall results verify the potential of the material and can be further used for the cartilage TE.

Characterization of Gelatin/Gellan Gum/Glycol Chitosan Ternary Hydrogel for Retinal Pigment Epithelial Tissue Reconstruction Materials.

The cellular transplantation approach to treat damaged or diseased retina is limited because of poor survival, distribution, and integration of cells after implantation to the sub-retinal space. To overcome this, it is important to develop a cell delivery system. In this study, a ternary hydrogel of gelatin (Ge)/gellan gum (GG)/glycol chitosan (CS) is suggested as a cell carrier for retinal tissue engineering (TE). Physicochemical properties such as porosity, swelling, sol fraction, and weight loss were measured. The mechanical study was performed with compressive strength and viscosity to confirm applicability in retinal TE. An in vitro experiment was carried out by encapsulating ARPE-19 in the designed hydrogel to measure viability and expression of retinal pigment epithelium-specific proteins and genes. The results showed that the ternary hydrogel system improves the mechanical properties and stability of the composite. Cell growth, survival, adhesion, and migration were enhanced as the CS was incorporated into the matrix. In particular, real-time polymerase chain reaction analysis showed a markedly improved specific gene expression rate in the Ge/GG/CS. Therefore, it is expected that the ternary system suggested in this study can be used as a promising material for retinal TE.

Role of substantia nigra-amygdala connections in surprise-induced enhancement of attention.

Coding of prediction error by midbrain dopamine neurons has been examined extensively in the framework of associative learning theory. Most of this research has focused on the role of prediction error in determining the reinforcement value of unconditioned stimuli: poorly predicted ("surprising") outcomes are more effective reinforcers and produce a greater dopamine response than well predicted outcomes. However, surprise also enhances attention to cues that signal poorly predicted outcomes. Previous reports from our laboratories demonstrated that circuitry, including the amygdala central nucleus (CeA), the cholinergic neurons of the substantia innominata/nucleus basalis region, and their innervation of the posterior parietal cortex, is critical to these surprise-induced enhancements of attention in associative learning. The present study considered the origin of prediction error information important for the operation of this system by examining the effects of disrupting communication between the midbrain substantia nigra pars compacta (SNc) and the CeA. Rats received unilateral lesions of the SNc and lesions of the CeA in either the contralateral or ipsilateral hemisphere. Contralateral lesions eliminated the surprise-induced enhancement of attention and learning that was displayed by rats with ipsilateral control lesions. These results show that SNc-CeA communication is critical to mechanisms by which the coding of prediction error by midbrain dopamine neurons is translated into enhancement of attention and learning modulated by the cholinergic system.