High-throughput laser printing of cells and biomaterials for tissue engineering

Abstract

In parallel with ink-jet printing and bioplotting, biological laser printing (BioLP) using laser-induced forward transfer has emerged as an alternative method in the assembly and micropatterning of biomaterials and cells. This paper presents results of high-throughput laser printing of a biopolymer (sodium alginate), biomaterials (nano-sized hydroxyapatite (HA) synthesized by wet precipitation) and human endothelial cells (EA.hy926), thus demonstrating the interest in this technique for three-dimensional tissue construction. A rapid prototyping workstation equipped with an IR pulsed laser (τ = 30 ns, λ = 1064 nm, f = 1–100 kHz), galvanometric mirrors (scanning speed up to 2000 mm s⁻¹) and micrometric translation stages (x, y, z) was set up. The droplet generation process was controlled by monitoring laser fluence, focalization conditions and writing speed, to take into account its mechanism, which is driven mainly by bubble dynamics. Droplets 70 μm in diameter and containing around five to seven living cells per droplet were obtained, thereby minimizing the dead volume of the hydrogel that surrounds the cells. In addition to cell transfer, the potential of using high-throughput BioLP for creating well-defined nano-sized HA patterns is demonstrated. Finally, bioprinting efficiency criteria (speed, volume, resolution, integrability) for the purpose of tissue engineering are discussed.

Introduction

Tissue architecture is complex, characterized by multiple cell types and matrix components precisely organized in three dimensions. Loss of tissue architecture due to trauma or disease leads to loss of tissue function. Since the late 1980s and the creation of the first workable definition of hybrid artificial organs [1], an increasing number of research groups throughout the world have begun to develop various novel tissue engineering approaches. As stated by Langer and Vacanti [2], these approaches apply the principles of engineering and life sciences to the development of biological substitutes which restore, maintain or improve tissue function or a whole organ. In other words, generating biological tissues in vitro involves the use of engineering and material methods, the appropriate combination of cells and the suitable biochemical and physicochemical factors to mimic both the microenvironment of cells and the microarchitecture of tissues in the body.

The basic idea underlying classical tissue engineering has been that the seeding of living cells onto a biocompatible and eventually biodegradable scaffold followed by the culturing of this system in a bioreactor would lead to the initial cell population expanding into a tissue. With an appropriate scaffold which mimics the biological extracellular matrix, it is expected that the developing tissue will adopt both the form and function of the desired organ, and would then be implanted into the recipient. Tremendous investigations have been realized in the synthesis and manufacture of biomaterials in order to obtain highly biocompatible and functional scaffolds.

In parallel with these approaches, some authors have suggested the building of three-dimensional (3D) biological structures by the technology of bioprinting: the automated, computer-aided deposition of cells, cell aggregates and biomaterials [3], [4]. To this end, commercially available inkjet printers have been successfully redesigned [5] or new ones built [6], [7] to pattern biological assemblies according to a computer-aided design template. Pressure-operated mechanical extruders such as bioplotters have also been developed to handle live cells and cell aggregates [8].

Parallel to these methods, laser-assisted printing technologies have emerged as an alternative method for the assembly and micropatterning of biomaterials and cells. Laser-guided direct writing (LGDW) is a technique capable of trapping multiple cells in a laser beam and depositing them as a steady stream onto arbitrary non-absorbing surfaces [10], [11]. Biological laser printing (BioLP) is based on the laser-induced forward-transfer (LIFT) technique in which a pulsed laser is used to induce the transfer of material from a source film spread onto an optically transparent quartz support to a substrate in close proximity to or in contact with the film [12]. Depending on whether or not the material is embedded in a laser light-absorbing matrix, a thin sacrificial absorbing layer is necessary. Accordingly, processes have been called matrix assisted pulsed laser evaporation-direct write (MAPLE-DW) [13] or absorbing film assisted-LIFT (AFA-LIFT) [14]. As BioLP has proved more effective with the aid of the intermediate light-absorbing layer, MAPLE-DW has been abandoned. Under suitable irradiation conditions, and for liquids presenting a wide range of rheologies, the material can be deposited in the form of well-defined circular droplets with a high degree of spatial resolution [9], [15].

Non-contact printing [16] is obtained through a jet formation which occurs, at a microsecond time scale, above a laser energy density threshold whose value depends on the rheological properties of liquid films and the thickness of the metallic absorbing layer [14]. By analogy with other studies in physics [17], [18], [19], it has been proposed that jet formation could be related to bubble dynamics [20]. Bubble growth depends mainly on viscosity and surface tension of the liquid, while bubble collapsing is related to the distance between the bubble front and the free surface [17]. Consequently, because droplet ejection is driven by bubble dynamics, high-throughput BioLP (HT-BioLP) requires spatial–temporal proximity between two pulses and, thus, two bubbles to be taken into account in order to avoid the perturbation of the collapsing of the initial bubble by another.

The present study develops a high-throughput workstation for printing different types of materials at micrometer resolution for the assembly of complex 3D biological structures. HT-BioLP parameters are first determined with regard to scanning or writing speed and bubble collapse-based mechanism. Then the potential of using BioLP to deposit a wide range of biological components was investigated, all of which are required for tissue engineering: biopolymers, nano-sized particles of HA as well as human endothelial cells. Finally, the potentiality of using HT-BioLP for tissue engineering is examined and discussed.