Fabien Guillemot

Laser assisted bioprinting of engineered tissue with high cell density and microscale organization

Over this decade, cell printing strategy has emerged as one of the promising approaches to organize cells in two and three dimensional engineered tissues. High resolution and high speed organization of cells are some of the key requirements for the successful fabrication of cell-containing two or three dimensional constructs. So far, none of the available cell printing technologies has shown an ability to concomitantly print cells at a cell-level resolution and at a kHz range speed. We have studied the effect of the viscosity of the bioink, laser energy, and laser printing speed on the resolution of cell printing. Accordingly, we demonstrate that a laser assisted cell printer can deposit cells with a microscale resolution, at a speed of 5 kHz and with computer assisted geometric control. We have successfully implemented such a cell printing precision to print miniaturized tissue like layouts with de novo high cell density and micro scale organization.

Cell patterning technologies for organotypic tissue fabrication

Bottom-up tissue engineering technologies address two of the main limitations of top-down tissue engineering approaches: the control of mass transfer and the fabrication of a controlled and functional histoarchitecture. These emerging technologies encompass mesoscale (e.g. cell sheets, cell-laden hydrogels and 3D printing) and microscale technologies (e.g. inkjet printing and laser-assisted bioprinting), which are used to manipulate and assemble cell-laden building blocks whose thicknesses correspond to the diffusion limit of metabolites, and present the capacity for cell patterning with microscale precision, respectively. Here, we review recent technological advances and further discuss how these technologies are complementary, and could therefore be combined for the biofabrication of organotypic tissues either in vitro, thus serving as realistic tissue models, or within a clinic setting.

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

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 (tau=30 ns, lambda=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 microm 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.

In vivo bioprinting for computer- and robotic-assisted medical intervention: preliminary study in mice

We present the first attempt to apply bioprinting technologies in the perspective of computer-assisted medical interventions. A workstation dedicated to high-throughput biological laser printing has been designed. Nano-hydroxyapatite (n-HA) was printed in the mouse calvaria defect model in vivo. Critical size bone defects were performed in OF-1 male mice calvaria with a 4 mm diameter trephine. Prior to laser printing experiments, the absence of inflammation due to laser irradiation onto mice dura mater was shown by means of magnetic resonance imaging. Procedures for in vivo bioprinting and results obtained using decalcified sections and x-ray microtomography are discussed. Although heterogeneous, these preliminary results demonstrate that in vivo bioprinting is possible. Bioprinting may prove to be helpful in the future for medical robotics and computer-assisted medical interventions.

Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells

Proper bone remodeling requires an active process of angiogenesis which in turn supplies the necessary growth factors and stem cells. This tissue cooperation suggests a cross‐talk between osteoblasts and endothelial cells. This work aims to identify the role of paracrine communication through vascular endothelial growth factor (VEGF) in co‐culture between osteoblastic and endothelial cells. Through a well defined direct contact co‐culture model between human osteoprogenitors (HOPs) and human umbilical vein endothelial cells (HUVECs), we observed that HUVECs were able to migrate along HOPs, inducing the formation of specific tubular‐like structures. VEGF165 gene expression was detected in the HOPs, was up‐regulated in the co‐cultured HOPs and both Flt‐1 and KDR gene expression increased in co‐cultured HUVECs. However, the cell rearrangement observed in co‐culture was promoted by a combination of soluble chemoattractive factors and not by VEGF165 alone. Despite having no observable effect on endothelial cell tubular‐like formation, VEGF appeared to have a crucial role in osteoblastic differentiation since the inhibition of its receptors reduced the co‐culture‐stimulated osteoblastic phenotype. This co‐culture system appears to enhance both primary angiogenesis events and osteoblastic differentiation, thus allowing for the development of new strategies in vascularized bone tissue engineering. J. Cell. Biochem. 106: 390–398, 2009.

Laser-assisted bioprinting for creating on-demand patterns of human osteoprogenitor cells and nano-hydroxyapatite.

Developing tools to reproduce and manipulate the cell micro-environment, including the location and shape of cell patterns, is essential for tissue engineering. Parallel to inkjet printing and pressure-operated mechanical extruders, laser-assisted bioprinting (LAB) has emerged as an alternative technology to fabricate two- and three-dimensional tissue engineering products. The objective of this work was to determine laser printing parameters for patterning and assembling nano-hydroxyapatite (nHA) and human osteoprogenitors (HOPs) in two and three dimensions with LAB. The LAB workstation used in this study comprised an infrared laser focused on a quartz ribbon that was coated with a thin absorbing layer of titanium and a layer of bioink. The scanning system, quartz ribbon and substrate were piloted by dedicated software, allowing the sequential printing of different biological materials into two and/or three dimensions. nHA printing material (bioink) was synthesized by chemical precipitation and was characterized prior and following printing using transmission electron microscopy, Fourier transformed infrared spectroscopy and x-ray diffraction. HOP bioink was prepared using a 30 million cells ml(-1) suspension in culture medium and cells were characterized after printing using a Live/Dead assay and osteoblastic phenotype markers (alcaline phosphatase and osteocalcin). The results revealed that LAB allows printing and organizing nHA and HOPs in two and three dimensions. LAB did not alter the physico-chemical properties of nHA, nor the viability, proliferation and phenotype of HOPs over time (up to 15 days). This study has demonstrated that LAB is a relevant method for patterning nHA and osteoblastic cells in 2D, and is also adapted to the bio-fabrication of 3D composite materials.

Laser-assisted cell printing: principle, physical parameters versus cell fate and perspectives in tissue engineering

We describe the physical parameters involved in laser-assisted cell printing and present evidence that this technology is coming of age. Finally we discuss how this high-throughput, high-resolution technique may help in reproducing local cell microenvironments, and thereby create functional tissue-engineered 3D constructs.

Recent advances in the design of titanium alloys for orthopedic applications

To increase an orthopedic implant’s lifetime, research trends have included the development of new titanium alloys made of nontoxic elements with suitable mechanical properties (low Young’s modulus – high fatigue strength), good workability and corrosion resistance. In accordance with the background on titanium and metallic biomaterials, recent interesting developments in titanium-based biomaterials are reported in this review, with a special emphasis on the design of new metastable β-titanium alloys for orthopedic applications. In addition, as the concept of titanium alloys can now be regarded as relatively old, having emerged at the beginning of the 1980s, the author suggests some future directions that would permit the emergence of a new generation of titanium implants.

Controlling laser-induced jet formation for bioprinting mesenchymal stem cells with high viability and high resolution.

Laser-assisted bioprinting is a versatile, non-contact, nozzle-free printing technique which has demonstrated high potential for cell printing with high resolution. Improving cell viability requires determining printing conditions which minimize shear stress for cells within the jet and cell impact at droplet landing. In this context, this study deals with laser-induced jet dynamics to determine conditions from which jets arise with minimum kinetic energies. The transition from a sub-threshold regime to jetting regime has been associated with a geometrical parameter (vertex angle) which can be harnessed to print mesenchymal stem cells with high viability using slow jet conditions. Finally, hydrodynamic jet stability is also studied for higher laser pulse energies which give rise to supersonic but turbulent jets.

Bioprinting by laser-induced forward transfer for tissue engineering applications: jet formation modeling

In this paper, a nanosecond LIFT process is analyzed both from experimental and modeling points of view. Experimental results are first presented and compared to simple estimates obtained from physical analysis, i.e. energy balance, jump relations and analytical pocket dynamics. Then a self-consistent 2D axisymmetric modeling strategy is presented. It is shown that data accessible from experiments, i.e. jet diameter and velocity, can be reproduced. Moreover, some specific mechanisms involved in the rear-surface deformation and jet formation may be described by some scales of hydrodynamic process, i.e. shock waves propagation and expansion waves, as a consequence of the laser heating. It shows that the LIFT process is essentially driven by hydrodynamics and thermal transfer, and that a coupled approach including self-consistent laser energy deposition, heating by thermal conduction and specific models for matter is required.