Raphaël Devillard

Cell patterning by laser-assisted bioprinting.

The aim of tissue engineering is to produce functional three-dimensional (3D) tissue substitutes. Regarding native organ and tissue complexity, cell density and cell spatial 3D organization, which influence cell behavior and fate, are key parameters in tissue engineering. Laser-Assisted Bioprinting (LAB) allows one to print cells and liquid materials with a cell- or picoliter-level resolution. Thus, LAB seems to be an emerging and promising technology to fabricate tissue-like structures that have the physiological functionality of their native counterparts. This technology has additional advantages such as automation, reproducibility, and high throughput. It makes LAB compatible with the (industrial) fabrication of 3D constructs of physiologically relevant sizes. Here we present exhaustively the numerous steps that allow printing of viable cells with a well-preserved micrometer pattern. To facilitate the understanding of the whole cell patterning experiment using LAB, it is discussed in two parts: (1) preprocessing: laser set-up, bio-ink cartridge and bio-paper preparation, and pattern design; and (2) processing: bio-ink printing on the bio-paper.

Stress-induced sphingolipid signaling: role of type-2 neutral sphingomyelinase in murine cell apoptosis and proliferation.

Background Sphingomyelin hydrolysis in response to stress-inducing agents, and subsequent ceramide generation, are implicated in various cellular responses, including apoptosis, inflammation and proliferation, depending on the nature of the different acidic or neutral sphingomyelinases. This study was carried out to investigate whether the neutral Mg2+-dependent neutral sphingomyelinase-2 (nSMase2) plays a role in the cellular signaling evoked by TNFalpha and oxidized LDLs, two stress-inducing agents, which are mitogenic at low concentrations and proapoptotic at higher concentrations. Methodology and Principal Findings For this purpose, we used nSMase2-deficient cells from homozygous fro/fro (fragilitas ossium) mice and nSMase2-deficient cells reconstituted with a V5-tagged nSMase2. We report that the genetic defect of nSMase2 (in fibroblasts from fro/fro mice) does not alter the TNFalpha and oxidized LDLs-mediated apoptotic response. Likewise, the hepatic toxicity of TNFalpha is similar in wild type and fro mice, thus is independent of nSMase2 activation. In contrast, the mitogenic response elicited by low concentrations of TNFalpha and oxidized LDLs (but not fetal calf serum) requires nSMase2 activation. Conclusion and Significance nSMase2 activation is not involved in apoptosis mediated by TNFalpha and oxidized LDLs in murine fibroblasts, and in the hepatotoxicity of TNFalpha in mice, but is required for the mitogenic response to stress-inducing agents.

Patterning of Endothelial Cells and Mesenchymal Stem Cells by Laser-Assisted Bioprinting to Study Cell Migration

Tissue engineering of large organs is currently limited by the lack of potent vascularization in vitro. Tissue-engineered bone grafts can be prevascularized in vitro using endothelial cells (ECs). The microvascular network architecture could be controlled by printing ECs following a specific pattern. Using laser-assisted bioprinting, we investigated the effect of distance between printed cell islets and the influence of coprinted mesenchymal cells on migration. When printed alone, ECs spread out evenly on the collagen hydrogel, regardless of the distance between cell islets. However, when printed in coculture with mesenchymal cells by laser-assisted bioprinting, they remained in the printed area. Therefore, the presence of mesenchymal cell is mandatory in order to create a pattern that will be conserved over time. This work describes an interesting approach to study cell migration that could be reproduced to study the effect of trophic factors.

Impression 3D en médecine régénératrice et ingénierie tissulaire

Additive manufacturing covers a number of fashionable technologies that attract the interest of researchers in biomaterials and tissue engineering. Additive manufacturing applied to regenerative medicine covers two main areas: 3D printing and biofabrication. If 3D printing has penetrated the world of regenerative medicine, bioassembly and bioimprinting are still in their infancy. The objective of this paper is to make a non-exhaustive review of these different complementary aspects of additive manufacturing in restorative and regenerative medicine or for tissue engineering.

Orofacial consequences of systemic sclerosis: A systematic review

Orofacial involvement is common and often understated in the treatment clinical guidelines of systemic sclerosis. It impairs daily life by having repercussions on comfort, nutrition, aesthetics and self-confidence. This review aimed at describing exhaustively the different orofacial consequences of systemic sclerosis. A systematic search was conducted using four databases (PubMed, Cochrane Library, Dentistry & Oral Sciences Source and SCOPUS) up to December 2016 according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses. Grey literature and hand search were also included. To be eligible for the inclusion, studies needed to meet the following criteria: randomised controlled trials, cross-sectional studies, case-control studies, pilot studies or cohort studies and full text available in English or French, with abstract. The studies had to concern at least 30 patients suffering from systemic sclerosis and having clinical and radiological oropharyngeal examination. The diagnosis of systemic sclerosis had to be determined according to precise recommendations; the retrieved oropharyngeal manifestations had to affect hard or soft tissues of the mouth and/or pharynx and needed to be evaluated with clinical measures. Study selection, risk bias assessment (Newcastle–Ottawa scale) and data extraction were performed by two independent reviewers. The retrieved features were microstomia and xerostomia associated with real hyposialia, temporomandibular joint symptoms, high caries experience, periodontal diseases as well as an increased risk of oral cavity and pharynx cancer. Early diagnosis enabling early management, prevention and oral hygiene is the key to avoid complicated and invasive procedures. Studies with higher level of evidence remain necessary to create standardised protocols.

Magnetic Resonance Imaging for tracking cellular patterns obtained by Laser-Assisted Bioprinting

Recent advances in the field of Tissue Engineering allowed to control the three-dimensional organization of engineered constructs. Cell pattern imaging and in vivo follow-up remain a major hurdle in in situ bioprinting onto deep tissues. Magnetic Resonance Imaging (MRI) associated with Micron-sized superParamagnetic Iron Oxide (MPIO) particles constitutes a non-invasive method for tracking cells in vivo. To date, no studies have utilized Cellular MRI as a tool to follow cell patterns obtained via bioprinting technologies. Laser-Assisted Bioprinting (LAB) has been increasingly recognized as a new and exciting addition to the bioprinting’s arsenal, due to its rapidity, precision and ability to print viable cells. This non-contact technology has been successfully used in recent in vivo applications. The aim of this study was to assess the methodology of tracking MPIO-labeled stem cells using MRI after organizing them by Laser-Assisted Bioprinting. Optimal MPIO concentrations for tracking bioprinted cells were determined. Accuracy of printed patterns was compared using MRI and confocal microscopy. Cell densities within the patterns and MRI signals were correlated. MRI enabled to detect cell patterns after in situ bioprinting onto a mouse calvarial defect. Results demonstrate that MRI combined with MPIO cell labeling is a valuable technique to track bioprinted cells in vitro and in animal models.

An easy-to-use and versatile method for building cell-laden microfibres

Fibre-shaped materials are useful for creating different functional three-dimensional (3D) structures that could mimic complex tissues. Several methods (e.g. extrusion, laminar flow or electrospinning) have been proposed for building hydrogel microfibres, with distinctive cell types and with different degrees of complexity. However, these methods require numerous protocol adaptations in order to achieve fibre fabricating and lack the ability to control microfibre alignment. Here, we present a simple method for the production of microfibers, based on a core shell approach, composed of calcium alginate and type I collagen. The process presented here allows the removal of the calcium alginate shell, after only 24 hours of culture, leading to stable and reproducible fibre shaped cellular constructs. With time of culture cells show to distribute preferentially to the surface of the fibre and display a uniform cellular orientation. Moreover, when cultured inside the fibres, murine bone marrow mesenchymal stem cells show the capacity to differentiate towards the osteoblastic lineage, under non-osteoinductive culture conditions. This work establishes a novel method for cellular fibre fabrication that due to its inherent simplicity can be easily upscaled and applied to other cell types.

Case Report of a Severe Recurrent Tongue Self-Injury in an Infant With Dystonia

Dystonia is characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive movements, postures, or both that are typically patterned, twisting, and sometimes tremulous. It is often initiated or worsened by voluntary action and associated with overflow muscle activation. In this article we report a case of severe oromandibular dystonia, which is a specific form of dystonia characterized by involuntary, action-induced tonic or clonic spasms of the masticatory, lingual, and pharyngeal musculature. Episodes of repeated tongue biting in a 17-month-old girl caused her to stay in the PICU for 4 weeks. These episodes were the consequence of dystonia induced by a perinatal stroke. We highlight the specific dental management that enabled us to treat the child without extractions. Facing this type of complex illness, we insist on the importance of interdisciplinary work with the goal of avoiding outdated techniques. The use of botulinum toxin seemed relevant.