JOURNAL OF COSMETIC SCIENCE 86 Despite recent advances and use of various technologies, the 3D printed skin lacks in dermal maturation and epidermal differentiation. Optimization of cell culture media providing maturogens and time management described as the fourth dimension need to be improved. Owning one of the most versatile skin equivalent model in terms of differ- ent skin cell types integrated yet, experience in skin functionality and LAB, we describe here the latest advances and experiments performed in our laboratories. FOUR-DIMENSIONAL LaBP: TECHNOLOGY AND BENEFITS TISSUE ENGINEERING EVOLUTIONS Tissue engineering evolved from the fi eld of biomaterials and medical devices. Conven- tional tissue engineering methods rely on the use of scaffolds to support and guide the subse- quent cellular and tissue organization (1–3). These top-down assembly approaches greatly rely on the self-organization of cells in response to environmental cues. They do not allow a fi ne control over the created fi nal structure and cell organization. On the contrary, bottom-up approaches, like additive fabrication technologies such as bioprinting, proceed by the assembly of small units which structure and organization can be fi nely tuned. Bioprinting offers the ability to create highly complex 3D architectures with living cells. Bioprinting methods have been developed to effectively and rapidly pattern living cells, biological macromolecules, and biomaterials. As a consequence, this cutting-edge technique has signifi cantly gained popularity and applicability in several fi elds as it facilitates physiologically relevant cell–cell and cell–matrix interactions allow- ing studies within an expected shorten time. BIOPRINTING Akin to ordinary ink printers, bioprinters have three major components to them. These are the hardware used, the type of bioink, and the material it is printed on (biomaterials). In bioprinting, there are three major types of printers that have been used. These are ink- jet, laser-assisted, and extrusion printers. Figure 1. Selected biofabrication approaches involving the use of hydrogels in form of so-called “bioink” (4).

NEW BIOPRINTED SKIN, COSMETIC IN VITRO MODEL 87 ▪ Laser-assisted bioprinters are less common and use lasers focused on an absorbing substrate to generate pressures that propel cell-containing materials onto a collector substrate. Printers that utilize lasers provide high-resolution printing and because it is a nozzle-free device, clogging of the nozzle is avoided (Figure 1, left). ▪ Thermal ink-jet printers electrically heat the printhead to produce air-pressure pulses that force droplets from the nozzle, whereas acoustic printers use pulses formed by piezoelectric or ultrasound pressure (Figure 1, middle). Ink-jet printers are mainly used in bioprinting for fast and large-scale products. ▪ Microextrusion printers use pneumatic or mechanical (piston or screw) dispensing systems to extrude continuous beads of material and/or cells (Figure 1, right). LABP TECHNOLOGY 3D laser-assisted bioprinter has a near infrared pulse laser source and a focus system to adjust the ejecta size. A laser is beamed through a transparent slide coated with an absorbent layer, enabling light energy to be converted into kinetic energy. A thin matrix layer, con- taining the component to be printed and a recipient substrate, is positioned a few microns away from the fi rst slide. Laser pulses are programmed to be sent approximately every nanosecond. This generates inkjets (cell containing mini-droplets), which are deposited layer by layer. In this system, physical ejection conditions—energy and viscosity—as well as droplet volume to around picoliter accuracy are controlled. The biological ink cartridge scans quickly, generating over 10,000 droplets a second with a resolution of 20 μm. Compared to man- ual skin equivalent production, the time to make a biological structure 1 cm2 and 200– 300 μm thick useful for in vitro testing is reduced by two-thirds. Our preliminary studies have shown that printable extracellular matrix and cells can be combined in a laser-assisted printing sequence to fabricate a stable and organized soft free form tissue, which can host a high cell density de novo. The LaBP can print versatile bio- logical patterns such as cell clusters, cell confl uent surface, and cell alignments according to computer-aided design. Also, a cell-level resolution of cell printing at a high speed (5 kHz) is achievable by this laser-assisted bioprinter. Such precision and speed were a prerequisite to apply the LaBP to cellularized tissue fabrication (5). As a matter of facts, several advantages have been associated to the use of 3D laser-assisted bioprinter (6) (Figure 2): ▪ Very high resolution compared to bioextrusion (single cell printing capability) ▪ Very high precision (μm) ▪ Very high cell viability compared to ink jet (nearly 100%) and ▪ Very high material viscosities possible use. However, the process is called four-dimensional (4D) bioprinting since it utilizes a fourth dimension: time. Once tissue is printed, the cells need time to communicate and self- assemble and this maturation is an important part of the biofabrication process. Indeed, the bioprinting of a 3D structure is not enough to create a functional tissue structure. Like more traditional scaffold-based methods, 4D bioprinting relies on self-organization capacities of cells and on morphogenetic processes. But unlike scaffold-based methods, bioprinting makes it possible to reproducibly control the initial 3D tissue structure. As