3D bioactive composite scaffolds for bone tissue engineering.

Three-dimensional bioprinting uses additive manufacturing techniques for the automated fabrication of hierarchically organized living constructs. The building blocks are often hydrogel-based bioinks, which need to be printed into structures with high shape fidelity to the intended computer-aided design. For optimal cell performance, relatively soft and printable inks are preferred, although these undergo significant deformation during the printing process, which may impair shape fidelity. While the concept of good or poor printability seems rather intuitive, its quantitative definition lacks consensus and depends on multiple rheological and chemical parameters of the ink. This review discusses qualitative and quantitative methodologies to evaluate printability of bioinks for extrusion- and lithography-based bioprinting. The physicochemical parameters influencing shape fidelity are discussed, together with their importance in establishing new models, predictive tools and printing methods that are deemed instrumental for the design of next-generation bioinks, and for reproducible comparison of their structural performance.

Printability and Shape Fidelity of Bioinks in 3D Bioprinting.

Manufacturing of hydrogel biomaterials with controlled mechanical properties for tissue engineering applications.

Three-dimensional bioprinting has emerged as a promising technique in tissue engineering applications through the precise deposition of cells and biomaterials in a layer-by-layer fashion. However, the limited availability of hydrogel bioinks is frequently cited as a major issue for the advancement of cell-based extrusion bioprinting technologies. It is well known that highly viscous materials maintain their structure better, but also have decreased cell viability due to the higher forces which are required for extrusion. However, little is known about the effect of the two distinct components of dynamic modulus of viscoelastic materials, storage modulus (G') and loss modulus (G″), on the printability of hydrogel-based bioinks. Additionally, 'printability' has been poorly defined in the literature, mostly consisting of gross qualitative measures which do not allow for direct comparison of bioinks. This study developed a framework for evaluating printability and investigated the effect of dynamic modulus, including storage modulus (G'), loss modulus (G″), and loss tangent (G″/G') on the printing outcome. Gelatin and alginate as model hydrogels were mixed at various concentrations to obtain hydrogel formulations with a wide range of storage and loss moduli. These formulations were then evaluated for the quantitatively defined values of extrudability, extrusion uniformity, and structural integrity. For extrudability, increasing either the loss or storage modulus increased the pressure required to extrude the bioink. A mathematical model relating the G' and G″ to the required extrusion pressure was derived based on the data. A lower loss tangent was correlated with increased structural integrity while a higher loss tangent correlated with increased extrusion uniformity. Gelatin-alginate composite hydrogels with a loss tangent in the range of 0.25-0.45 exhibited an excellent compromise between structural integrity and extrusion uniformity. In addition to the characterization of a common bioink, the methodology introduced in this paper could also be used to evaluate the printability of other bioinks in the future.

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Optimization of gelatin-alginate composite bioink printability using rheological parameters: a systematic approach.

The dawn of 3D printing in medicals has pinned the domain with hopes of vitality in many patients combating with multitude of diseases. Also entitled as Bio-printing, this is appertained to its sequential printing of precursor ink, embodying cells and polymer/composite, in predetermined trajectory. Precursor ink, in addition to cells, constitutes predominantly hydrogels ascribed to its biodegradability and mimic ability of body's anatomy and mechanical features, e.g. bones, etc. This review paper is devoted to explicate bio-printing (3D/4D) of alginate hydrogels, which are the extract from brown algae, through extrusion additive manufacturing. Alginates are salt derivative of alginic acid and constitute long chain of polysaccharides, which furnishes pliability and gelling adeptness to its structure. Alginate hydrogel (employed for extrusion) can be pristine or composite relying on the requisite properties (target application controlled or in-vivo environment) e.g. Alginate-natural (gelatin/agarose/collagen/hyaluronic acid/etc.) and Alginate-synthetic (polyethylene glycol (PEG)/pluronic F127/etc.). Extrusion additive manufacturing of Alginate is preponderate among others with its uncomplicated processing, material efficiency (cut down on wastage), and outspread adaptability for viscosities (0.03-6*104 Pa.s) but the procedure is limited by resolution(200 ?m) in addition to accuracy. However, 3D-fabricated bio-structures display rigidness (unvarying with conditions) that lacks smart response which is reassured by accounting time feature as noteworthy accessory to printing, interpreting as 4D Bio-printing. This review propounds specific processing itinerary for alginate (meanwhile traversing across its composites/blends with natural and synthetic consideration) in extrusion along with its pre-/during/post-processing parameters intrinsic to process. Furthermore, propensity is also presented in its (Alginate extrusion processing) application for tissue engineering, i.e. bones, cartilage (joints), brain (neural), ear, heart (cardiac), eyes (corneal), etc. due to worldwide quandary over accessibility to natural organs for diverse kinds of diseases. Additionally, the review contemplates recently invented advance printing i.e. 4D printing for biotic species with its challenges and future opportunities.

https://iopscience.iop.org/article/10.1088/1758-5090/ab331e/pdf

Review of alginate-based hydrogel bioprinting for application in tissue engineering.

Different bioprinting techniques have been used to produce cell-laden alginate hydrogel structures, however these approaches have been limited to 2D or simple three-dimension (3D) structures. In this study, a new extrusion based bioprinting technique was developed to produce more complex alginate hydrogel structures. This was achieved by dividing the alginate hydrogel cross-linking process into three stages: primary calcium ion cross-linking for printability of the gel, secondary calcium cross-linking for rigidity of the alginate hydrogel immediately after printing and tertiary barium ion cross-linking for long-term stability of the alginate hydrogel in culture medium. Simple 3D structures including tubes were first printed to ensure the feasibility of the bioprinting technique and then complex 3D structures such as branched vascular structures were successfully printed. The static stiffness of the alginate hydrogel after printing was 20.18 ± 1.62 KPa which was rigid enough to sustain the integrity of the complex 3D alginate hydrogel structure during the printing. The addition of 60 mM barium chloride was found to significantly extend the stability of the cross-linked alginate hydrogel from 3 d to beyond 11 d without compromising the cellular viability. The results based on cell bioprinting suggested that viability of U87-MG cells was 93 ± 0.9% immediately after bioprinting and cell viability maintained above 88% ± 4.3% in the alginate hydrogel over the period of 11 d.

https://iopscience.iop.org/article/10.1088/1758-5090/7/4/045012/pdf

Three-dimensional bioprinting of complex cell laden alginate hydrogel structures

Glycogen Synthase Kinase-3 (GSK3 or GSK-3) is a promiscuous protein kinase and its phosphorylation of its diverse substrates has major influences on many areas of physiology and pathology, including cellular metabolism, lineage commitment and neuroscience. GSK3 was one of the first identified substrates of the heavily studied oncogenic kinase AKT, phosphorylation by which inhibits GSK3 activity via the formation of an autoinhibitory pseudosubstrate sequence. This has led to investigation of the role of GSK3 inhibition as a key component of the cellular responses to growth factors and insulin, which stimulate the class I PI 3-Kinases and in turn AKT activity and GSK3 phosphorylation. GSK3 has been shown to phosphorylate several upstream and downstream components of the PI3K/AKT/mTOR signalling network, including AKT itself, RICTOR, TSC1 and 2, PTEN and IRS1 and 2, with the potential to apply feedback control within the network. However, it has been clear for some time that functionally distinct, insulated pools of GSK3 exist which are regulated independently, so that for some GSK3 substrates such as β-catenin, phosphorylation by GSK3 is not controlled by input from PI3K and AKT. Instead, as almost all GSK3 substrates require a priming phosphorylated residue to be 4 amino acids C-terminal to the Ser/Thr phosphorylated by GSK3, the predominant form of regulation of the activity of GSK3 often appears to be through control over these priming events, specific to individual substrates. Therefore, a major role of GSK3 can be viewed as an amplifier of the electrostatic effects on protein function which are caused by these priming phosphorylation events. Here we discuss these different aspects to GSK3 regulation and function, and the functions of GSK3 as it integrates with signalling through the PI3K-AKT-mTOR signalling axis.

https://pure.hw.ac.uk/ws/files/15594631/1_s2.0_S2212492617301240_main.pdf

GSK3 and its interactions with the PI3K/AKT/mTOR signalling network

Loss of function of the PTEN tumour suppressor, resulting in dysregulated activation of the phosphoinositide 3-kinase (PI3K) signalling network, is recognized as one of the most common driving events in prostate cancer development. The observed mechanisms of PTEN loss are diverse, but both homozygous and heterozygous genomic deletions including PTEN are frequent, and often accompanied by loss of detectable protein as assessed by immunohistochemistry (IHC). The occurrence of PTEN loss is highest in aggressive metastatic disease and this has driven the development of PTEN as a prognostic biomarker, either alone or in combination with other factors, to distinguish indolent tumours from those likely to progress. Here, we discuss these factors and the consequences of PTEN loss, in the context of its role as a lipid phosphatase, as well as current efforts to use available inhibitors of specific components of the PI3K/PTEN/TOR signalling network in prostate cancer treatment.

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Prostate cancer, PI3K, PTEN and prognosis.

In many human cell types, the class I phosphoinositide 3-kinases play key roles in the control of diverse cellular processes including growth, proliferation, survival and polarity. This is achieved through their activation by many cell surface receptors, leading to the synthesis of the phosphoinositide lipid signal, PIP3, which in turn influences the function of numerous direct PIP3-binding proteins. Here we review PI3K pathway biology and analyse the evolutionary distribution of its components and their functions. The broad phylogenetic distribution of class I PI3Ks in metazoa, amoebozoa and choannoflagellates, implies that these enzymes evolved in single celled organisms and were later co-opted into metazoan intercellular communication. A similar distribution is evident for the AKT and Cytohesin groups of downstream PIP3-binding proteins, with other effectors and pathway components appearing to evolve later. The genomic and functional phylogeny of regulatory systems such as the PI3K pathway provides a framework to improve our understanding of the mechanisms by which key cellular processes are controlled in humans.

https://pure.hw.ac.uk/ws/files/8963952/class.pdf

Class I PI 3-kinases: Function and evolution.

Three dimensional (3D) bioprinting of multiple cell types within optimised extracellular matrices has the potential to more closely model the 3D environment of human physiology and disease than current alternatives. In this study, we used a multi-nozzle extrusion bioprinter to establish models of glioblastoma made up of cancer and stromal cells printed within matrices comprised of alginate modified with RGDS cell adhesion peptides, hyaluronic acid and collagen-1. Methods were developed using U87MG glioblastoma cells and MM6 monocyte/macrophages, whilst more disease relevant constructs contained glioblastoma stem cells (GSCs), co-printed with glioma associated stromal cells (GASCs) and microglia. Printing parameters were optimised to promote cell-cell interaction, avoiding the 'caging in' of cells due to overly dense cross-linking. Such printing had a negligible effect on cell viability, and cells retained robust metabolic activity and proliferation. Alginate gels allowed the rapid recovery of printed cell protein and RNA, and fluorescent reporters provided analysis of protein kinase activation at the single cell level within printed constructs. GSCs showed more resistance to chemotherapeutic drugs in 3D printed tumour constructs compared to 2D monolayer cultures, reflecting the clinical situation. In summary, a novel 3D bioprinting strategy is developed which allows control over the spatial organisation of tumour constructs for pre-clinical drug sensitivity testing and studies of the tumour microenvironment.

https://pure.hw.ac.uk/ws/files/25729281/1_s2.0_S221249261930079X_main_1_.pdf

Miguel A. Hermida

Papers

Three-dimensional bioprinting of complex cell laden alginate hydrogel structures

GSK3 and its interactions with the PI3K/AKT/mTOR signalling network

Prostate cancer, PI3K, PTEN and prognosis.

Class I PI 3-kinases: Function and evolution.

Three dimensional in vitro models of cancer: Bioprinting multilineage glioblastoma models.