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.

Human amniotic membrane for guided bone regeneration of calvarial defects in mice.

Due to its biological properties, human amniotic membrane (hAM) is widely studied in the field of tissue engineering and regenerative medicine. hAM is already very attractive for wound healing and it may be helpful as a support for bone regeneration. However, few studies assessed its potential for guided bone regeneration (GBR). The purpose of the present study was to assess the potential of the hAM as a membrane for GBR. In vitro, cell viability in fresh and cryopreserved hAM was assessed. In vivo, we evaluated the impact of fresh versus cryopreserved hAM, using both the epithelial or the mesenchymal layer facing the defect, on bone regeneration in a critical calvarial bone defect in mice. Then, the efficacy of cryopreserved hAM associated with a bone substitute was compared to a collagen membrane currently used for GBR. In vitro, no statistical difference was observed between the conditions concerning cell viability. Without graft material, cryopreserved hAM induced more bone formation when the mesenchymal layer covered the defect compared to the defect left empty. When associated with a bone substitute, such improved bone repair was not observed. These preliminary results suggest that cryopreserved hAM has a limited potential for GBR.

Effect of BMP-2 from matrices of different stiffnesses for the modulation of stem cell fate.

Stem cells cultured on extracellular matrix (ECM) with different stiffnesses have been shown to engage into different lineage commitments. However, in vivo, the components of the ECM are known to bind and strongly interact with growth factors. The effect, on the stem cell fate, of the cooperation between the mechanical properties and the growth factor in the same microenvironment has not yet been investigated. Here, we propose a protocol for mimicking this stem cell microenvironment with an in vitro system. This system consists in grafting (without using a spacer) biomolecules that contain N-termini groups onto hydrogel (poly(acrylamide-co-acrylic acid)) surfaces of various stiffnesses ranging from 0.5 to 70 kPa. First, we demonstrate that the commitment of mesenchymal stem cell populations changes in response to the substrate's rigidity, with myogenic differentiation occurring at 13-17 kPa and osteogenic differentiation at 45-49 kPa. Chemical grafting of soft and stiff matrices with an osteogenic factor (BMP-2(mimetic peptide)) results only in osteogenic differentiation. Also, when grafted on even softer gels (0.5-3.5 kPa), the BMP-2(mimetic peptide) had no effect on the stem cell differentiation. We prove that correct organization of F-actin cytoskeleton due to the mechanical properties of the microenvironment is necessary for BMP-induced smad1/5/8 phosphorylation and nuclear translocation. These results suggest that stem cell differentiation is dictated mechanically, but in the presence of a biochemical factor, the effect of the mechanical factor on stem cell commitment is modified. This can explain the diversity of stem cell behaviors in vivo where different growth factors are sequestrated on the ECM.