Alginate-gelatin Hydrogel Research Articles

Interactions between gelatin and sodium alginate: UV and FTIR studies

The interaction between macromolecular chains of gelatin and anionic polysaccharide sodium alginate during the formation of polyelectrolyte complexes was examined using UV and FTIR spectroscopy. It has been shown that in an aqueous phase, where the pH is slightly higher than the pI of gelatin, complexes are formed through hydrogen bonds as well as electrostatic interactions between two biopolymers. The complex formation is accompanied by a change in the secondary structure of gelatin. At a low weight (w/w) ratio of polysaccharide to gelatin (up to 0.2) a decrease in the share of collagen-like helices and an increase in the share of the random coil conformation of gelatin chains were reported. SEM was used to identify a fundamental distinction between the morphologies of the gelatin hydrogel and the mixed alginate–gelatin hydrogel with a low mass ratio of biopolymers. Spherical formations were found in the structure of the latter that were interpreted as aggregations of complexes.

Study of hybrid alginate/gelatin hydrogel-incorporated niosomal Aloe vera capable of sustained release of Aloe vera as potential skin wound dressing

Nowadays, wound dressings serve as advanced skin products, which mainly aim to accelerate the wound healing process. The wound dressings with a potential of localized and prolonged release of therapeutic agents have attracted tremendous attention. In this study, synthesis of a sustained release of niosomal Aloe vera (AV) loaded in alginate/gelatin (AG) hybrid hydrogel is aimed at improving skin regeneration as wound dressing. For this purpose, AV-loaded niosomes are synthesized and incorporated in the hybrid AG hydrogel. The size and polydispersity index (PdI) of niosomes, AV entrapment efficacy and AV in vitro release are characterized. In addition, the hydrogel characteristic, such as swelling ratio, degradation behavior and mechanical property, are studied. MTT assay is utilized to evaluate the effect of AV incorporation and release on the proliferation of fibroblast cells. Results demonstrate that size, PdI and EE% of AV-loaded niosomes are 270.080 nm, 0.108 and 42.039 ± 4.090%, respectively, and in vitro release of AV is about 20% after 7 days. AG hybrid hydrogel loaded by niosomal AV shows an extended sustained release manner, where its swelling ratio and percentage of degradation are 60 wt% after 72 h and 70 wt% after 6 days, respectively. The average Young’s modulus of the hybrid hydrogel is measured around 12.64 ± 1.3 kPa, which seems suitable as a wound dressing. Finally, MTT assay confesses an increased fibroblast proliferation in the presence of AV, particularly in the niosomal AV-loaded hydrogel. Concludingly, alginate/gelation hybrid hydrogel incorporated with niosomal AV, with sustained release potential can be suggested as a promising candidate for wound dressing applications.

Nanoceria-loaded injectable hydrogels for potential age-related macular degeneration treatment.

The major purpose of this article is to evaluate oligochitosan coated cerium oxide nanoparticles (OCCNPs) alginate laden injectable hydrogels and their potential treatment for age-related macular degeneration (AMD). The water soluble OCCNPs were loaded within injectable hydrogels as antioxidative agents. The release of OCCNPs from hydrogel, radical scavenging properties, and biocompatibility were evaluated and calculated in vitro. The effects of OCCNP laden hydrogel downregulating expression of angiogenic proteins and proinflammatory cytokines were quantified in human retinal pigment epithlium-19 (ARPE-19) and umbilical endothelium cell lines. The hydrogels behaved with moderate swelling and controllable degradation. The laden OCCNPs were released in a controlled manner in vitro during two months of testing. The OCCNP loaded hydrogels exhibited robust antioxidative properties in oxygen radical absorbance capacity tests and reduced apoptosis in H2 O2 -induced ARPE-19 cells. Furthermore, OCCNP loaded injectable hydrogels are biocompatible and suppressed the ipopolysaccharides-induced inflammation response in ARPE-19 cells, and inhibited expression of vascular endothelium growth factor in human ARPE-19 and umbilical endothelium cell lines. The alginate-gelatin injectable hydrogel loaded OCCNPs are biocompatible and have high potential in protecting cells from apoptosis, angiogenesis, and production of proinflammatory cytokines in AMD cellular models. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2795-2804, 2018.

3D bioprinted rat Schwann cell-laden structures with shape flexibility and enhanced nerve growth factor expression.

Three-dimensional (3D) bioprinting composite alginate-gelatin hydrogel has encouraged the fabrication of cell-laden functional structures with cells from various tissues. However, reports focusing on printing this hydrogel for nerve tissue research are limited. This study aims at building in vitro Schwann cell 3D microenvironment with customized shapes through 3D bioprinting technology. Rat Schwann cell RSC96s encapsulated in composite alginate-gelatin hydrogel were printed with an extrusion-based bioprinter. Cells maintained high viability of 85.35 ± 6.19% immediately after printing and the printed hydrogel supported long-term Schwann cell proliferation for 2weeks. Furthermore, after 14days of culturing, Schwann cells cultured in printed structures maintained viability of 92.34 ± 2.19% and showed enhanced capability of nerve growth factor (NGF) release (142.41 ± 8.99pg/ml) compared with cells from two-dimensional culture (92.27 ± 9.30pg/ml). Specific Schwann cell marker S100β was also expressed by cells in printed structures. These printed structures may have the potential to be used as in vitro neurotrophic factor carriers and could be integrated into complex biomimetic artificial structures with the assistance of 3D bioprinting technology.

Controlling alginate oxidation conditions for making alginate-gelatin hydrogels

In the present work, we discuss how oxidation conditions can affect the physical properties of oxidized alginate and crosslinking it with gelatin. We show that the amount of aldehyde groups produced on oxidized alginate backbone increases by increasing alginate concentration even in constant molar ratio of sodium periodate to alginate's repeating units. Increasing the concentration of alginate solution, promote the extent of chain scission and decreases the molecular weight of oxidized alginate, which can be due to the increased possibility of molecular collisions and oxidizing two adjacent uronic acids in the chain. By changing the oxidation condition, therefore, we can produce oxidized alginate with same degree of oxidation but different molecular weights, which change the sol-gel state of alginate-gelatin mixtures. Using oxidized alginates with different molecular weights, we suggest that the aldehyde end groups in alginate chains have the dominant effect in crosslinking with gelatin.

Tuning Alginate-Gelatin Bioink Properties by Varying Solvent and Their Impact on Stem Cell Behavior

Bioink optimization is considered as one of main challenges in cell-laden 3D bioprinting. Alginate-Gelatin (Alg-Gel) hydrogel have been extensively used as bioink. However, its properties could be influenced by various parameters, and little is known about the evidence featuring the impact of solvent. Here we investigated four Alg-Gel bioink by varying solvent ionic strength (named B-1, B-2, B-3 and B-4). Mechanical properties and printability of bioink samples and their impacts on behaviors of encapsulated epidermal stem cells (ESCs) were tested. Bioink with increased ionic strength of solvent showed decreased stiffness and viscosity, and increased swelling and degradation by printability and mechanical property tests. Due to the increased swelling and degradation was associated with shape-maintenance of post-printing constructs, B-3 and B-4 were hardly observable after 14 days. Cellular behaviors were assessed through viability, proliferation, aggregation and differentiation tests. B-2 with optimal properties resulted in higher viability and proliferation of ESCs, and further facilitated cellular aggregation and lineage differentiation. We demonstrated that the solvent can be tuned by ionic strength to control the properties of Alg-Gel bioink and post-printing constructs, which represented a promising avenue for promotion of therapeutic stem cell behaviors in 3D bioprinting.

Fabrication of micropatterned alginate-gelatin and k-carrageenan hydrogels of defined shapes using simple wax mould method as a platform for stem cell/induced Pluripotent Stem Cells (iPSC) culture

Micropatterning techniques involve soft lithography, which is laborious, expensive and restricted to a narrow spectrum of biomaterials. In this work we report, first time employment of patterned wax moulds for generation of micropatterned alginate-gelatin and κ-carrageenan (κ-CRG) hydrogel systems by a novel, simple and cost effective method. We generated and characterized uniform and reproducible micropatterned hydrogels of varying sizes and shapes such as square projections, square grooves, and circular grids and crisscrossed hillocks. The rheological analysis showed that κ-carrageenan hydrogels had higher gel strength when compared to alginate-gelatin hydrogels. Human Mesenchymal stem cells (hMSCs) and Human Induced Pluripotent Stem Cells (hiPSCs) were found to be cytocompatible with these hydrogels. This micropatterned hydrogel system may have potential application in tissue engineering and also in understanding the basic biology behind the stem cell/iPSC fate.

Mechanical behaviour of alginate-gelatin hydrogels for 3D bioprinting.

Hydrogels comprised of alginate and gelatin have demonstrated potential as biomaterials in three dimensional (3D) bioprinting applications. However, as with all hydrogel-based biomaterials used in extrusion-based bioprinting, many parameters influence their performance and there is limited data characterising the behaviour of alginate-gelatin (Alg-Gel) hydrogels. Here we investigated nine Alg-Gel blends by varying the individual constituent concentrations. We tested samples for printability and print accuracy, compressive behaviour and change over time, and viability of encapsulated mesenchymal stem cells in bioprinted constructs. Printability tests showed a decrease in strand width with increasing concentrations of Alg-Gel. However due to the increased viscosity associated with the higher Alg-Gel concentrations, the minimum width was found to be 0.32mm before blends became too viscous to print. Similarly, printing accuracy was increased in higher concentrations, exceeding 90% in some blends. Mechanical properties were assessed through uniaxial compression testing and it was found that increasing concentrations of both Alg and Gel resulted in higher compressive modulus. We also deemed 15min crosslinking in calcium chloride to be sufficient. From our data, we propose a blend of 7%Alg-8%Gel that yields high printability, mechanical strength and stiffness, and cell viability. However, we found the compressive behaviour of Alg-Gel to reduce rapidly over time and especially when incubated at 37°C. Here we have reported relevant data on Alg-Gel hydrogels for bioprinting. We tested for biomaterial properties and show that these hydrogels have many desirable characteristics that are highly tunable. Though further work is needed before practical use in vivo can be achieved.

Oxidized Alginate-Gelatin Hydrogel: A Favorable Matrix for Growth and Osteogenic Differentiation of Adipose-Derived Stem Cells in 3D.

Alginate-based hydrogels are extensively used matrices for cell encapsulation, but they need to be modified to recapitulate chemical, microstructural, and mechanical properties of the native extracellular matrix. Like other cell types, mesenchymal stem cells exhibit rounded and clustered morphologies when they are embedded in alginate hydrogels. In this study, we use covalently cross-linked oxidized alginate-gelatin hydrogels to encapsulate human adipose-derived stem cells in order to investigate cell growth, viability, and morphology during osteogenic differentiation taking advantage of the different physicochemical properties of this modified alginate-based hydrogel in comparison to those of the pristine alginate hydrogel. We investigate the effect of hydrogel compositions on stem cell behavior in 3D. Higher viability and the spreading morphology of encapsulated cells with interconnected networks were observed in high gelatin containing compositions. More filopodial protrusions from multicellular nodules were noticed during osteogenic differentiation in the hydrogels having a high amount of gelatin, confirming their suitability for cell encapsulation and bone tissue engineering applications.

Abstract 2072: Combination of CDODA-Me, a glycyrrhetinic acid derivative, and Erlotinib overcomes chemo-resistance in NSCLC PDX spheroids and 3D bio-printed cells

Abstract Patient-derived Xenografts (PDXs) are considered as relevant preclinical model for anticancer drug development due to original recapitulation of patient genetic profile, gene expression patterns and tissue histology. In this study, we investigated combination efficacy of CDODA-Me (Methyl 2-cyano-3,11-dioxo-18-olean-1,12-dien-30-oate) and TKI inhibitor Erlotinib (ERL) against Lung NSCLC PDX spheroids and 3D bio-printed PDX cells. NSCLC PDX cells (EGFR T790M mutants) were obtained from Dr. Rishi’s Laboratory. PDX spheroids were grown in DMEM/ F12 media supplemented with L-glutamine, B27 supplement, recombinant human epidermal growth factor (EGF) and recombinant human basic fibroblast growth factor (bFGF). Spheroids were treated with CDODA-Me, ERL alone and in combination. Cell viability was measured by MTT assay. Western blot analysis was used to study the modulation of Bcl-xL, MDR1 and ABCG2 in treated PDX spheroids. For 3D bio-printing of PDX cells, hydrogels were prepared by partial cross-linking of sodium alginate (4.5% w/v) and gelatin (1% w/v) mixture with 40mM CaCl2 solution. PDX Cells were mixed with partially cross-linked hydrogel and printed with Inkredible 3D bio-printer (CELLINK, Sweden). Bio-printed scaffolds were fully cross-linked by 160 mM CaCl2 solution and then incubated overnight with cell culture media. The scaffolds were treated with CDODA-Me and ERL alone and in combination. After 48 h cell viabilities were determined by live/dead assay using fluorescence microscopy. MTT assay showed that approximately 65% and 74% viability was observed at 10 µM ERL and 2.5 µM CDODA-Me respectively. Decreased spheroid cell viability was observed in ERL and CDODA-Me combination treatment. Our western blot studies showed down-regulation of Bcl-xL, MDR1 and ABCG2 in combination group. Further, 81.04 ± 5.65, 78.65 ± 3.98 and 74.35 ± 4.24 percent viable PDX cells were observed in the bio-printed scaffolds after 48, 72 and 96 h respectively. Higher percentage of dead cells (52.62 ± 1.66) were found in the combination group than CDODA-Me (28.39 ± 1.60) and ERL (29.62 ± 4.91) alone. In conclusion, CDODA-Me in combination with ERL was found to be effective against human lung PDX spheroids and bio-printed PDX cells by decreasing the cell viability and overcoming drug resistance. Partially cross-linked sodium alginate-gelatin hydrogel enhances the possibility of PDX cell bio-printing with high cell survival rate. CDODA-Me can be considered as an effective neo-adjuvant to improve ERL efficacy in human NSCLC. Citation Format: Arindam Mondal, Aragaw Gebeyehu, Ebony Nottingham, Arvind Bagde, Subramanian Ramakrishnan, Arun K. Rishi, Mandip Singh. Combination of CDODA-Me, a glycyrrhetinic acid derivative, and Erlotinib overcomes chemo-resistance in NSCLC PDX spheroids and 3D bio-printed cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2072. doi:10.1158/1538-7445.AM2017-2072

Bioprinting multidimensional constructs: a quantitative approach to understanding printed cell density and redistribution phenomena

Currently, bioprinting high cellular fractions in bio-inks represents a critical step towards engineered tissue models with enhanced biological relevance. However, precise spatiotemporal mapping of high cell densities within hydrogel materials represents a significant challenge. To address these needs and challenges, this paper advances a systematic multidimensional analytical framework to quantitatively determine the spatial variations in cell density and temporal evolution of cell redistribution phenomena within bioprinted cell-laden fiber-based constructs as a function of the complex interplay of the various processing parameters for an alginate-gelatin hydrogel material system. For a one dimensional analysis, fibers printed at high relative viscosities exhibit large contact angles in the cross-sectional plane with a high degree of cell dispersion (Dd = 96.8 ± 6.27%). Conversely, fibers printed at low relative viscosities exhibit small contact angles with cells coalescing towards the fiber midline (Dd = 76.3 ± 4.58%). The observed in-process redistribution of cells is attributed to the flow topology inside the needle tip. The precise mechanism is governed by the temperature-dependent viscoplastic material properties as demonstrated numerically using the Herschel–Bulkley model. In the 2D analysis, the printed grid structures yield differences in local cell densities between the strut and cross regions within the fibers. At low relative viscosities, cells aggregate in regions where two overlapping fibers fuse together, yielding a high cell density ratio (1.79 ± 0.04) between the cross and strut regions. However, at high relative viscosities, the cell density ratio decreases (0.96 ± 0.03). In the 3D analysis, due to gravity and extrusion process effects, initially uniform bio-ink cell distributions within bio-inks are redistributed as a function of printing time, yielding quantitative increases in cell density for the initial layers, followed by quantitative decreases in the subsequent layers. Finally, a time course incubation study of the 3D bioprinted cell-laden construct reveals a time-dependent increase in cell density owing to proliferation that accelerates the material degradation.

Designing Porous Bone Tissue Engineering Scaffolds with Enhanced Mechanical Properties from Composite Hydrogels Composed of Modified Alginate, Gelatin, and Bioactive Glass.

The combination of biodegradable polymers and bioactive inorganic materials is being widely used for designing bone tissue engineering scaffolds. Here we report a composite hydrogel system composed of bioactive glass incorporated in covalently cross-linked oxidized alginate-gelatin hydrogel (ADA-GEL) for designing porous scaffolds with tunable stiffness and degradability using freeze-drying technique. Because of the presence of bioactive glass, the cross-linking kinetic and cross-linking degree of the hydrogels are significantly increased, which is the main factor for the measured enhanced mechanical strength of the bioactive glass containing ADA-GEL scaffolds. The hydrogels with high cross-linking degree exhibit low protein release profile and low degradability. Apatite formation on bioactive glass containing hydrogel-based scaffolds is confirmed by FTIR. Bone marrow-derived stromal cell growth is promoted in pristine ADA-GEL and 1% bioactive glass containing ADA-GEL scaffolds compared to the scaffolds of pure alginate, alginate-gelatin blended hydrogel, and 5% bioactive glass containing ADA-GEL. Initial studies indicated that the scaffolds, especially without bioactive glass, support osteogenic differentiation of murine bone marrow stromal cell line in the absence of foreign osteogenic stimulating supplements; however, they exhibit low levels of osteogenic expression.

Macromolecular interactions in alginate–gelatin hydrogels regulate the behavior of human fibroblasts

Due to the presence of tripeptide arginine–glycine–aspartic acid, gelatin is considered a very promising additive material to improve the cytocompatibility of alginate-based hydrogels. Two different strategies, physical blending and chemical crosslinking with gelatin, are used in this study to modify alginate hydrogel. As the intermolecular interactions between the polysaccharide and protein in the resulting physically blended and chemically crosslinked hydrogels are different, significant differences in the properties of these hydrogel types, regarding especially their surface topography, degradation kinetics, mechanical properties, and protein release behavior, are observed. Cellular behavior on both types of alginate–gelatin hydrogels is investigated using primary human dermal fibroblasts to elucidate the effects of the different structural, mechanical, and degradation properties of the produced hydrogels on fibroblast attachment and growth. The hydrogel that is chemically crosslinked with gelatin exhibits the highest degree of cytocompatibility regarding adhesion, proliferation, metabolic activity, and morphology of growing fibroblasts.

Design, fabrication and characterization of oxidized alginate–gelatin hydrogels for muscle tissue engineering applications

In this study, we reported the preparation of self cross-linked oxidized alginate-gelatin hydrogels for muscle tissue engineering. The effect of oxidation degree (OD) and oxidized alginate/gelatin (OA/GEL) weight ratio were examined and the results showed that in the constant OA/GEL weight ratio, both cross-linking density and Young's modulus enhanced by increasing OD due to increment of aldehyde groups. Furthermore, the degradation rate was increased with increasing OD probably due to decrement in alginate molecular weight during oxidation reaction facilitated degradation of alginate chains. MTT cytotoxicity assays performed on Wharton's Jelly-derived umbilical cord mesenchymal stem cells cultured on hydrogels with OD of 30% showed that the highest rate of cell proliferation belong to hydrogel with OA/GEL weight ratio of 30/70. Overall, it can be concluded from all obtained results that the prepared hydrogel with OA/GEL weight ratio and OD of 30/70 and 30%, respectively, could be proper candidate for use in muscle tissue engineering.

3D Bioprinting of complex channels-Effects of material, orientation, geometry, and cell embedding.

Creating filled or hollow channels within 3D tissues has become increasingly important in tissue engineering. Channels can serve as vasculature enhancing medium perfusion or as conduits for nerve regeneration. The 3D biofabrication seems to be a promising method to generate these structures within 3D constructs layer-by-layer. In this study, geometry and interface of bioprinted channels were investigated with micro-computed tomography and fluorescent imaging. In filament printing, size and shape of printed channels are influenced by their orientation, which was analyzed by printing horizontally and vertically aligned channels, and by the ink, which was evaluated by comparing channels printed with an alginate-gelatin hydrogel or with an emulsion. The influence of geometry and cell-embedding in the hydrogel on feature size and shape was investigated by printing more complex channels. The generation of hollow channels, induced through leaching of a support phase, was monitored over time. Horizontally aligned channels provided 16× smaller cross-sectional areas than channels in vertical orientation. The smallest feature size of hydrogel filaments was twice as large compared to emulsion filaments. Feature size and shape depended on the geometry but did not alter when living cells were embedded. With that knowledge, channels can be consciously tailored to the particular needs.

Topical Astragaloside IV-Releasing Hydrogel Improves Healing of Skin Wounds <i>in Vivo</i>

Topical delivery of therapeutic agents at the time of injury to accelerate skin repair and prevent the formation of scars during the wound healing process has received increasing attention and represents a novel regenerative and prophylactic strategy for wound treatment. The aim of this study was to invesigate, for the first time, the influence of topical astragaloside IV-releasing hydrogel on the wound repair and regeneration. Using the sodium alginate-gelatin as a hydrogel vehicle, the astragaloside IV was incorporated into the topical carrier and kept releasing with a sustained manner at the wound site. With the rat skin excision model, regulation of the astragaloside IV hydrogel on the wound repair and regeneration were investigated. It was found that the astragaloside IV hydrogel was effective in the skin wound repair, leading to a significant improvement on the wound closure, collagen synthesis and skin tensile strength recovery. Meanwhile, for the first time, that functions of astragaloside IV hydrogel in activating the skin appendages regeneration and increasing the transforming growth factor-β(1) (TGF-β(1)) level in serum were shown. Results of this study provided evidence for the alginate-gelatin hydrogel as efficient carrier for the topical delivery of bioactive molecules to the injured site. The astragaloside IV releasing hydrogel was shown a promising therapeutic formulation for wound healing, as well as its regenerative feature and underlying mechanism contribute to the skin regeneration were disclaimed.