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Current Advancements and Future Perspectives of 3d Tissue Printing

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Introduction

Tissue engineering’s singular aim is to authentically replicate native tissue, in terms of architecture and functionality. The comprehensive understanding of past technological advances and current challenges in the field of 3D tissue printing is essential to surpass the restricted clinical applications of this method of tissue engineering. The need for a reliable fabrication of tissues originates from the issues linked to current treatments. Organ transplants are analogous with long term complications from surgery, immune rejection, complex administrative approvals, Tissue fabrication/engineering is a clinical solution which not only accommodates for organ donor shortages, but provides a solution to numerous medical issues: providing 3D cellular matrices for clinical testing, …. etc.

The Multifaceted Applications of 3D Tissue Printing

The emergence of new technologies, in combination with a better understanding of biochemical processes has led to the development of 3D bio-printing. This form of tissue engineering, enables a more systematic fabrication of tissues, a superior control over tissue geometry, and the easier combination of cells and materials. However, the further development of these biotechnologies is still needed to overcome the limited clinical applications of this method. (clinical limits of this process)In this review, we first assess the strategic approaches taken in 3D printing a tissue, to then examine and compare 3D tissue printing methods, 3D Bioprinting Approaches Biomimicry. A biomimetic approach to tissue printing aims to apply biological understanding at the microscale to reproduce a functional tissue. A substantial knowledge base of the microenvironment, native biochemical cues, cellular mechanics, functional and supporting cell types, extracellular matrix (ECM) composition is indispensable to successfully imitate and replicate elements of native tissue. However, this approach has shown to be successful in Examples of this technique, Self-assembly, Mini-tissues, 3D Bioprinters. The principal technologies used by tissue engineers for the deposition and patterning of biological materials are inkjet printers, microextrusion printers, and laser-assisted printers. The significant features which are used to compare these bioprinting strategies are their cell viability, the surface resolution a printer produces, and the biomaterials which the printer can use. Inkjet Inkjet, or drop-on-demand printers, are the most commonly used types of 3D printers for both biological and non-biological applications, due to their availability, low cost, and high speed.

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The inkjet printer replicates a digital pattern of a desired 3D substrate to form a structure through the repeated deposition of droplets at predesignated locations onto an elevator stage. The location of the droplet deposition relative to the x and y axes is controlled by the printhead, whereas the formation of the substrate along the z axis is controlled by the elevator stage. The bio-ink, a mixture of cells and biomaterials, is loaded into the printhead, and extruded from the nozzle, depending on the printer either through thermal or acoustic force. In a thermal inkjet printing process small air bubbles are formed within the electrically-heated printhead, which then collapse. The resulting pressure pulses induce the ejection of bio-ink droplets out of the nozzle to a predefined location onto the substrate. The volume of the drop, ranging from 10 to 150 pL, is tuned as a function of applied temperature gradient, frequency of pulse, and viscosity of the bioink.

The localised heating within the bio-ink chamber of thermal printers ranges from 200 to 300 C. However, multiple studies have revealed overall temperatures to only rise by a maximum of 4 to 10 C, due to the short duration of the heating (~ 2µs). As such, the rise in temperature was demonstrated to have no significant effect on the stability of biomaterials, biological elements, such as DNA molecules, or on the viability and post-printing function of mammalian cells. The thermal inkjet printer is frequently employed for its high print speed, low cost, wide availability and high resolution potential (20 – 100 μ).

However, the exposure of cells and materials to thermal and mechanical stress, low droplet directionality, non-uniform droplet size, frequent clogging of the nozzle and unreliable cell encapsulation all contribute to the drawbacks of applying this technique to tissue formation. Cell microencapsulation is an essential tool for tissue engineers to bypass immune rejection. The encapsulation of cells within a permeable membrane, commonly made from a soft polymer or hydrogel, enables the exchange of nutrients, growth factors and waste products with surrounding tissues, whilst segregating cells from the body’s immune system. This process is therefore essential to circumvent the adverse use of immuno-suppressant drugs, and for the development of a successful substitute tissue or organ. The piezoelectric inkjet printer is equipped with a piezoelectric crystal which under applied voltage undergoes a rapid shape change and generates an acoustic wave within the printhead. The acoustic wave separates the bioink into many droplets, which are then ejected at regular intervals from the nozzle. Other forms of acoustic inkjet printers utilise ultrasound to control the size of the liquid droplets, by adjusting the pulse, duration and amplitude of the ultrasound field. O

ne study combined two low cost and flexible cell patterning methods, Acoustic Droplet Ejection (ADE) and Acqueous Two Phase Systems (ATPSs) to create a contact-less, and nozzle-less process. The cells are patterned in an acqueous open pool environment and then collected by a destination plate placed on the path of the cells; avoiding the damaging impact of mechanical and sheer stresses on cell viability. Relative to thermal inkjet printers, acoustic printers generate a more uniform droplet size and ejection directionality, and reduce cellular exposure to thermal and pressure stresses. In addition to these advantages, acoustic inkjet printers may be equipped with multiple ejectors, permitting the simultaneous printing of multiple cells and biomaterials.

However, the frequencies at which piezoelectric printers function, ranging from 15 to 25 kHz, remain a principal concern in generating cell lysis or impairment. Overall, the use of inkjet printers for tissue engineering presents various advantages, such as short preparation time, cost-effectiveness, high resolution (less than 1 pL) and high speed (1-10,000 droplets/second); all of which originate from an uncomplicated design and software, and widely available components. However, inkjet printers require the bioink to be in liquid state when extruded from the nozzle, which will in turn form a solid 3D structure when printed. This challenge was addressed in a 2010 study by Skardal et al. , in which layers of hydrogel were crosslinked following printing onto the plate through UV irradiation. Crosslinking of the biomaterial post-printing may be done through chemical, pH or ultraviolet means, however this process decreases the viability and functionality of the cells, and increases the printing time. An additional drawback of inkjet printing is that the viscosity of the bioink must remain “ideally below 10 centipoise” or the excessive force necessary to eject a droplet would hinder cell viability. Lastly, the bioink of an inkjet printer often contains low cell concentrations as higher cell concentrations may inhibit crosslinking mechanisms, and to reduce shear stress, avoid nozzle clogging and facilitate droplet formation.

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