Development of alginate dialdehyde-gelatin based bioink with methylcellulose for improving printability.

This study used methylcellulose (MC) to improve the printability of the alginate dialdehyde-gelatin (ADA-GEL) based bioink. The printability as well as the capability to maintain shape fidelity of ADA-GEL could be enhanced by the addition of 9% (w/v) MC. Moreover, the properties of the ink crosslinked with Ca2+ and Ba2+ were investigated. The samples crosslinked with Ba2+ were more stable and stiffer than the Ca2+ crosslinked samples. However, both Ca2+ and Ba2+ crosslinked samples exhibited a similar trend of MC release during incubation under cell culture conditions. The toxicity test indicated that both samples (crosslinked with Ca2+ and Ba2+) exhibited no toxic potential. The fabrication of cell-laden constructs using the developed bioinks was evaluated. The viability of ST2 cells in Ba2+ crosslinked samples increased while for Ca2+ crosslinked samples, a decreased viability was observed over the incubation time. After 21 days, cell spreading in the hydrogels crosslinked with Ba2+ occurred. However, a certain degree of cell damage was observed after incorporating the cells in the high viscosity bioink.

Rational Design of a Triple-Layered Coaxial Extruder System: in silico and in vitro Evaluations Directed Toward Optimizing Cell Viability.

Biofabrication is a rapidly evolving field whose main goal is the manufacturing of three-dimensional (3D) cell-laden constructs that closely mimic tissues and organs. Despite recent advances on materials and techniques directed toward the achievement of this goal, several aspects such as tissue vascularization and prolonged cell functionality are limiting bench-to-bedside translation. Extrusion-based 3D bioprinting has been devised as a promising biofabrication technology to overcome these limitations, due to its versatility and wide availability. Here, we report the development of a triple-layered coaxial nozzle for use in the biomanufacturing of vascular networks and vessels. The design of the coaxial nozzle was first optimized toward guaranteeing high cell viability upon extrusion. This was done with the aid of in silico evaluations and their subsequent experimental validation by investigating the bioprinting of an alginate-based bioink. Results confirmed that the values for pressure distribution predicted by in silico experiments resulted in cell viabilities above 70% and further demonstrated the effect of layer thickness and extrusion pressure on cell viability. Our work paves the way for the rational design of multi-layered coaxial extrusion systems to be used in biofabrication approaches to replicate the very complex structures found in native organs and tissues.

Cell-laden alginate dialdehyde-gelatin hydrogels formed in 3D printed sacrificial gel.

Alginate dialdehyde-gelatin (ADA-GEL) hydrogels have been reported to be suitable matrices for cell encapsulation. In general, application of ADA-GEL as bioink has been limited to planar structures due to its low viscosity. In this work, ring shaped constructs of ADA-GEL hydrogel were fabricated by casting the hydrogel into sacrificial molds which were 3D printed from 9% methylcellulose and 5% gelatin. Dissolution of the supporting structure was observed during the 1st week of sample incubation. In addition, the effect of different crosslinkers (Ba2+ and Ca2+) on the physicochemical properties of ADA-GEL and on the behavior of encapsulated MG-63 cells was investigated. It was found that Ba2+ crosslinked network had more than twice higher storage modulus, and mass decrease to 70% during incubation compared to 42% in case of hydrogels crosslinked with Ca2+. In addition, faster increase in cell viability during incubation and earlier cell network formation were observed after Ba2+ crosslinking. No negative effects on cell activity due to the use of sacrificial materials were observed. The approach presented here could be further developed for cell-laden ADA-GEL bioink printing into complex 3D structures.

Biofabrication and Characterization of Alginate Dialdehyde-Gelatin Microcapsules Incorporating Bioactive Glass for Cell Delivery Application.

The effect of the incorporation of 45S5 bioactive glass (BG) microparticles (mean particle size ≈ 2 µm) on the fabrication and physicochemical properties of alginate dialdehyde-gelatin hydrogel capsules is investigated. The addition of BG particles decreases the hydrogel gelation time by ≈79% and 91% for the samples containing 0.1% w/v and 0.5% w/v BG, respectively. Moreover, it results in increasing average diameter of hydrogel capsules produced via a pressure-driven extrusion technique from about 1000 µm for the samples without BG to about 1700 and 1900 µm for the samples containing BG at concentrations of 0.1% w/v and 0.5% w/v, respectively. The presence of BG particles in the capsules decreases the degradation rate and improves the bioactivity of the materials. The viability of MG-63 cells encapsulated in all samples increases during the first 7 d of cultivation and maintains the same level during 21 d of cultivation. The early cell viability in samples containing BG is lower than that in samples without BG. The results show that 45S5 BG can positively regulate the osteogenic activity of cells incorporated in hydrogel capsules. The fabricated composite capsules exhibit promising potential for cell delivery in bone regeneration applications.

Oxidized alginate hydrogels with the GHK peptide enhance cord blood mesenchymal stem cell osteogenesis: A paradigm for metabolomics-based evaluation of biomaterial design.

Oxidized alginate hydrogels are appealing alternatives to natural alginate due to their favourable biodegradability profiles and capacity to self-crosslink with amine containing molecules facilitating functionalization with extracellular matrix cues, which enable modulation of stem cell fate, achieve highly viable 3-D cultures, and promote cell growth. Stem cell metabolism is at the core of cellular fate (proliferation, differentiation, death) and metabolomics provides global metabolic signatures representative of cellular status, being able to accurately identify the quality of stem cell differentiation. Herein, umbilical cord blood mesenchymal stem cells (UCB MSCs) were encapsulated in novel oxidized alginate hydrogels functionalized with the glycine-histidine-lysine (GHK) peptide and differentiated towards the osteoblastic lineage. The ADA-GHK hydrogels significantly improved osteogenic differentiation compared to gelatin-containing control hydrogels, as demonstrated by gene expression, alkaline phosphatase activity and bone extracellular matrix deposition. Metabolomics revealed the high degree of metabolic heterogeneity in the gelatin-containing control hydrogels, captured the enhanced osteogenic differentiation in the ADA-GHK hydrogels, confirmed the similar metabolism between differentiated cells and primary osteoblasts, and elucidated the metabolic mechanism responsible for the function of GHK. Our results suggest a novel paradigm for metabolomics-guided biomaterial design and robust stem cell bioprocessing. STATEMENT OF SIGNIFICANCE: Producing high quality engineered bone grafts is important for the treatment of critical sized bone defects. Robust and sensitive techniques are required for quality assessment of tissue-engineered constructs, which result to the selection of optimal biomaterials for bone graft development. Herein, we present a new use of metabolomics signatures in guiding the development of novel oxidised alginate-based hydrogels with umbilical cord blood mesenchymal stem cells and the glycine-histidine-lysine peptide, demonstrating that GHK induces stem cell osteogenic differentiation. Metabolomics signatures captured the enhanced osteogenesis in GHK hydrogels, confirmed the metabolic similarity between differentiated cells and primary osteoblasts, and elucidated the metabolic mechanism responsible for the function of GHK. In conclusion, our results suggest a new paradigm of metabolomics-driven design of biomaterials.

Cell laden alginate-keratin based composite microcapsules containing bioactive glass for tissue engineering applications.

Microcapsules based on alginate-keratin, alginate dialdehyde (ADA)-keratin and ADA-keratin-45S5 bioactive glass (BG) were successfully prepared. The samples were characterized by light microscopy, scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The results showed that ADA-based materials possess higher degradation rate compared to alginate-based materials. The incorporation of BG particles (mean particle size: 2.0 µm) improved the bioactivity of the materials. Moreover, the biological properties of the samples were evaluated by encapsulating MG-63 osteosarcoma cells into the microcapsules. The cell viability in all samples increased during 21 days of cultivation. However, the presence of 0.5% BG particle seemed to have initial negative effect on cell growth compared to other samples without BG. On the other hand, the positive effect of CaP formation was visible after 3 weeks in the BG containing samples. The results are relevant to consider the development of cell laden bioinks incorporating inorganic bioactive particles for biofabrication approaches.

Oxidized Alginate-Based Hydrogels for Tissue Engineering Applications: A Review.

Oxidized alginate (OA)-based hydrogels have drawn considerable attention as biodegradable materials for tissue engineering applications. OA possesses a faster degradation rate and contains more reactive groups compared to native alginate. This review summarizes the research publications reporting the development of OA-based hydrogels for tissue engineering applications including bone, cartilage, blood vessel, cornea, and other soft tissues, highlighting OA key properties and processing approaches.

Cell specificity of magnetic cell seeding approach to hydrogel colonization.

Tissue-engineered scaffolds require an effective colonization with cells. Superparamagnetic iron oxide nanoparticles (SPIONs) can enhance cell adhesion to matrices by magnetic cell seeding. We investigated the possibility of improving cell attachment and growth on different alginate-based hydrogels using fibroblasts and endothelial cells (ECs) loaded with SPIONs. Hydrogels containing pure alginate (Alg), alginate dialdehyde crosslinked with gelatin (ADA-G) and Alg blended with G or silk fibroin (SF) were prepared. Endothelial cells and fibroblasts loaded with SPIONs were seeded and grown on hydrogels for up to 7 days, in the presence of magnetic field during the first 24 h. Cell morphology (fluorescent staining) and metabolic activity (WST-8 assay) of magnetically-seeded versus conventionally seeded cells were compared. Magnetic seeding of ECs improved their initial attachment and further growth on Alg/G hydrogel surfaces. However, we did not achieve an efficient and stable colonization of ADA-G films with ECs even with magnetic cell seeding. Fibroblast showed good initial colonization and growth on ADA-G and on Alg/SF. This effect was further significantly enhanced by magnetic cell seeding. On pure Alg, initial attachment and spreading of magnetically-seeded cells was dramatically improved compared to conventionally-seeded cells, but the effect was transient and diminished gradually with the cessation of magnetic force. Our results demonstrate that magnetic seeding improves the strength and uniformity of initial cell attachment to hydrogel surface in cell-specific manner, which may play a decisive role for the outcome in tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2948-2956, 2017.