Bioactive gelatin-sheets as novel biopapers to support prevascularization organized by laser-assisted bioprinting for bone tissue engineering.

Despite significant advances in the management of patients with oral cancer, maxillofacial reconstruction after ablative surgery remains a clinical challenge. In bone tissue engineering, biofabrication strategies have been proposed as promising alternatives to solve issues associated with current therapies and to produce bone substitutes that mimic both the structure and function of native bone. Among them, laser-assisted bioprinting (LAB) has emerged as a relevant biofabrication method to print living cells and biomaterials with micrometric resolution onto a receiving substrate, also called 'biopaper'. Recent studies have demonstrated the benefits of prevascularization using LAB to promote vascularization and bone regeneration, but mechanical and biological optimization of the biopaper are needed. The aim of this study was to apply gelatin-sheet fabrication process to the development of a novel biopaper able to support prevascularization organized by LAB for bone tissue engineering applications. Gelatin-based sheets incorporating bioactive glasses (BGs) were produced using various freezing methods and crosslinking (CL) parameters. The different formulations were characterized in terms of microstructural, physical, mechanical, and biological properties in monoculture and coculture. Based on multi-criteria analysis, a rank scoring method was used to identify the most relevant formulations. The selected biopaper underwent additional characterization regarding its ability to support mineralization and vasculogenesis, its bioactivity potential andin vivodegradability. The biopaper 'Gel5wt% BG1wt%-slow freezing-CL160 °C 24 h' was selected as the best candidate, due to its suitable properties including high porosity (91.69 ± 1.55%), swelling ratio (91.61 ± 0.60%), Young modulus (3.97 × 104± 0.97 × 104Pa) but also its great cytocompatibility, osteogenesis and bioactivity properties. The preorganization of human umbilical vein endothelial cell using LAB onto this new biopaper led to the formation of microvascular networks. This biopaper was also shown to be compatible with 3D-molding and 3D-stacking strategies. This work allowed the development of a novel biopaper adapted to LAB with great potential for vascularized bone biofabrication.

Description of a multidisciplinary model of care in a French cohort of adult patients with tuberous sclerosis complex.

BACKGROUND: Tuberous sclerosis complex (TSC) is a rare autosomal dominant genetic disorder. Due to the various manifestations of TSC and their potential complications, a multidisciplinary care approach is recommended by consensus guidelines. OBJECTIVES: Our study aimed to give a complete description of our TSC adult cohort and to evaluate the multidisciplinary and interdisciplinary management model. METHODS: Data on each adult patient diagnosed with TSC, including disease manifestations, interventions and outcomes, were collected at baseline and updated annually. A multidisciplinary TSC approach with all the recommended explorations was carried out annually. RESULTS: 90 patients were enrolled in Centre Hospitalier Universitaire de Bordeaux, between January 2000 and September 2018. Median age of patients at inclusion was 37 years (range, 27-47) and 20 years old at diagnosis of TSC. Regarding the occurrence of TSC manifestations, 97% of the patients had cutaneous lesions, 89% had neurological manifestations, 83% had renal manifestations and 100% had dental lesions with pits. More than half the patients had sclerotic bone lesions (68%), TSC-associated neuropsychiatric disorders (64%) and lymphangioleiomyomatosis (59%). A TSC multidisciplinary approach was developed including a global follow-up and an evaluation of TSC targeting organs, according to the recommendations. A satisfaction survey revealed global and entire satisfaction of patients with TSC. CONCLUSION: We obtained an accurate description of a cohort of adult patients with TSC. Our multidisciplinary approach model allowed us to provide optimal management of patients with TSC with a high level of patient satisfaction.

Laser-Assisted Bioprinting for Bone Repair.

Bioprinting is a novel technological approach that has the potential to solve unmet questions in the field of tissue engineering. Laser-assisted bioprinting (LAB), due to its unprecedented cell printing resolution and precision, is an attractive tool for the in situ printing of a bone substitute. Here, we describe the protocol for LAB and its use for the in situ bioprinting of mesenchymal stromal cells, associated with collagen and nanohydroxyapatite, in order to favor bone regeneration in a calvaria defect model in mice.

Towards an in vitro model of the glomerular barrier unit with an innovative bioassembly method.

BACKGROUND: The development of an artificial glomerular unit may be pivotal for renal pathophysiology studies at a multicellular scale. Using a tissue engineering approach, we aimed to reproduce in part the specific glomerular barrier architecture by manufacturing a glomerular microfibre (Mf). METHODS: Immortalized human glomerular cell lines of endothelial cells (GEnCs) and podocytes were used. Cells and a three-dimensional (3D) matrix were characterized by immunofluorescence with confocal analysis, Western blot and polymerase chain reaction. Optical and electron microscopy were used to study Mf and cell shapes. We also analysed cell viability and cell metabolism within the 3D construct at 14 days. RESULTS: Using the Mf manufacturing method, we repeatedly obtained a cellularized Mf sorting human glomerular cells in 3D. Around a central structure made of collagen I, we obtained an internal layer composed of GEnC, a newly formed glomerular basement membrane rich in α5 collagen IV and an external layer of podocytes. The cell concentration, optimal seeding time and role of physical stresses were modulated to obtain the Mf. Cell viability and expression of specific proteins (nephrin, synaptopodin, vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrandt factor (vWF)) were maintained for 19 days in the Mf system. Mf ultrastructure, observed with EM, had similarities with the human glomerular barrier. CONCLUSION: In summary, with our 3D bio-engineered glomerular fibre, GEnC and podocytes produced a glomerular basement membrane. In the future, this glomerular Mf will allow us to study cell interactions in a 3D system and increase our knowledge of glomerular pathophysiology.

Layer-by-layer bioassembly of poly(lactic) acid membranes loaded with coculture of HBMSCs and EPCs improves vascularization in vivo.

Layer-by-layer (LBL) BioAssembly method was developed to enhance the control of cell distribution within 3D scaffolds for tissue engineering applications. The objective of this study was to evaluate in vivo the development of blood vessels within LBL bioassembled membranes seeded with human primary cells, and to compare it to cellularized massive scaffolds. Poly(lactic) acid (PLA) membranes fabricated by fused deposition modeling were seeded with monocultures of human bone marrow stromal cells or with cocultures of these cells and endothelial progenitor cells. Then, four cellularized membranes were assembled in LBL constructs. Early osteoblastic and endothelial cell differentiation markers, alkaline phosphatase, and von Willebrand's factor, were expressed in all layers of assemblies in homogenous manner. The same kind of LBL assemblies as well as cellularized massive scaffolds was implanted subcutaneously in mice. Human cells were observed in all scaffolds seeded with cells, but not in the inner parts of massive scaffolds. There were significantly more blood vessels observed in LBL bioassemblies seeded with cocultures compared to all other samples. LBL bioassembly of PLA membranes seeded with a coculture of human cells is an efficient method to obtain homogenous cell distribution and blood vessel formation within the entire volume of a 3D composite scaffold.

Micropatterning of endothelial cells to create a capillary-like network with defined architecture by laser-assisted bioprinting.

Development of a microvasculature into tissue-engineered bone substitutes represents a current challenge. Seeding of endothelial cells in an appropriate environment can give rise to a capillary-like network to enhance prevascularization of bone substitutes. Advances in biofabrication techniques, such as bioprinting, could allow to precisely define a pattern of endothelial cells onto a biomaterial suitable for in vivo applications. The aim of this study was to produce a microvascular network following a defined pattern and preserve it while preparing the surface to print another layer of endothelial cells. We first optimise the bioink cell concentration and laser printing parameters and then develop a method to allow endothelial cells to survive between two collagen layers. Laser-assisted bioprinting (LAB) was used to pattern lines of tdTomato-labeled endothelial cells cocultured with mesenchymal stem cells seeded onto a collagen hydrogel. Formation of capillary-like structures was dependent on a sufficient local density of endothelial cells. Overlay of the pattern with collagen I hydrogel containing vascular endothelial growth factor (VEGF) allowed capillary-like structures formation and preservation of the printed pattern over time. Results indicate that laser-assisted bioprinting is a valuable technique to pre-organize endothelial cells into high cell density pattern in order to create a vascular network with defined architecture in tissue-engineered constructs based on collagen hydrogel.

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.

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.

Layer-by-layer bioassembly of cellularized polylactic acid porous membranes for bone tissue engineering.

The conventional tissue engineering is based on seeding of macroporous scaffold on its surface ("top-down" approach). The main limitation is poor cell viability in the middle of the scaffold due to poor diffusion of oxygen and nutrients and insufficient vascularization. Layer-by-Layer (LBL) bioassembly is based on "bottom-up" approach, which considers assembly of small cellularized blocks. The aim of this work was to evaluate proliferation and differentiation of human bone marrow stromal cells (HBMSCs) and endothelial progenitor cells (EPCs) in two and three dimensions (2D, 3D) using a LBL assembly of polylactic acid (PLA) scaffolds fabricated by 3D printing. 2D experiments have shown maintain of cell viability on PLA, especially when a co-cuture system was used, as well as adequate morphology of seeded cells. Early osteoblastic and endothelial differentiations were observed and cell proliferation was increased after 7 days of culture. In 3D, cell migration was observed between layers of LBL constructs, as well as an osteoblastic differentiation. These results indicate that LBL assembly of PLA layers could be suitable for BTE, in order to promote homogenous cell distribution inside the scaffold and gene expression specific to the cells implanted in the case of co-culture system.

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.

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 Mg(2+)-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.

Resveratrol inhibits the mTOR mitogenic signaling evoked by oxidized LDL in smooth muscle cells.

OBJECTIVES: Smooth muscle cell (SMC) proliferation is a major feature in atherosclerosis, since it contributes to the formation of the fibrous cap, thus to plaque stability, but also to arterial stenosis and post-angioplasty restenosis. Among the various mitogenic signaling pathways involved in SMC proliferation, the mTOR pathway regulates both the cell cycle and cell growth. Resveratrol, a polyphenolic compound from grapes and red wine, has potential anti-atherogenic and anti-cancer properties. This work was designed to investigate the activation of the mTOR pathway by the proatherogenic oxidized LDL (oxLDL) in SMC, and the potential inhibitory effect of resveratrol. RESULTS: mTOR and its downstream target p70S6 kinase are phosphorylated and activated by mitogenic concentrations of oxLDL (50 microg/ml), and are involved in SMC proliferation, as assessed by the inhibitory effect of the mTOR inhibitor rapamycin. The activation of mTOR signaling by oxLDL, requires the upstream activation of PI3K and Akt, as assessed by the inhibitory effect of the PI3K inhibitor Ly294002 on mTOR activation and DNA synthesis. Resveratrol blocked the oxLDL-induced phosphorylation and activation of the PI3K/Akt/mTOR/p70S6K pathway and strongly inhibited both the DNA synthesis and proliferation of SMC. This activity is independent of the anti-oxidant effect and of AMPK activation by resveratrol. CONCLUSION: These data indicate that the mTOR pathway is activated by oxLDL via PI3K/PDK1/Akt, and is required for SMC proliferation. Resveratrol blocks specifically this pathway, thereby inhibiting oxLDL-induced SMC proliferation. These data highlight a new property for resveratrol that could contribute to the general anti-atherogenic properties of this polyphenol.