N.C. Rivron

Vascularization in tissue engineering

Tissue engineering has been an active field of research for several decades now. However, the amount of clinical applications in the field of tissue engineering is still limited. One of the current limitations of tissue engineering is its inability to provide sufficient blood supply in the initial phase after implantation. Insufficient vascularization can lead to improper cell integration or cell death in tissue-engineered constructs. This review will discuss the advantages and limitations of recent strategies aimed at enhancing the vascularization of tissue-engineered constructs. We will illustrate that combining the efforts of different research lines might be necessary to obtain optimal results in the field.

Spheroid culture as a tool for creating 3D complex tissues

3D cell culture methods confer a high degree of clinical and biological relevance to in vitro models. This is specifically the case with the spheroid culture, where a small aggregate of cells grows free of foreign materials. In spheroid cultures, cells secrete the extracellular matrix (ECM) in which they reside, and they can interact with cells from their original microenvironment. The value of spheroid cultures is increasing quickly due to novel microfabricated platforms amenable to high-throughput screening (HTS) and advances in cell culture. Here, we review new possibilities that combine the strengths of spheroid culture with new microenvironment fabrication methods that allow for the creation of large numbers of highly reproducible, complex tissues.

Tissue assembly and organization: Developmental mechanisms in microfabricated tissues

In vitro-generated tissues hold significant promise in modern biology since they can potentially mimic physiological and pathological tissues. However, these are currently structurally and functionally of limited complexity and necessitate self-organization and recapitulation of tissue development mechanisms in vitro. Tools derived from nano- and microfabrications along with bottom-up strategies are emerging to allow the fabrication of primitive tissues structures that can remodel overtime. Subsequently, clues are accumulating to show that, beyond genetic material, both intrinsic tissue architectures and microenvironmental cues can lead to morphogenesis related mechanisms in vitro. The question arises, however, as how we may design and assemble structures prone to adequate tissue remodeling, predict and manipulate those developmental mechanisms in vitro? Systems integrating architectural, physical and molecular cues will allow more systematic investigation of basic principles of tissue morphogenesis, differentiation or maintenance and will feedback to reproduce the dynamic of tissue development in vitro and form more complex tissues.

Tissue deformation spatially modulates VEGF signaling and angiogenesis

Physical forces play a major role in the organization of developing tissues. During vascular development, physical forces originating from a fluid phase or from cells pulling on their environment can alter cellular signaling and the behavior of cells. Here, we observe how tissue deformation spatially modulates angiogenic signals and angiogenesis. Using soft lithographic templates, we assemble three-dimensional, geometric tissues. The tissues contract autonomously, change shape stereotypically and form patterns of vascular structures in regions of high deformations. We show that this emergence correlates with the formation of a long-range gradient of Vascular Endothelial Growth Factor (VEGF) in interstitial cells, the local overexpression of the corresponding receptor VEGF receptor 2 (VEGFR-2) and local differences in endothelial cells proliferation. We suggest that tissue contractility and deformation can induce the formation of gradients of angiogenic microenvironments which could contribute to the long-range patterning of the vascular system.

Sonic Hedgehog-activated engineered blood vessels enhance bone tissue formation

Large bone defects naturally regenerate via a highly vascularized tissue which progressively remodels into cartilage and bone. Current approaches in bone tissue engineering are restricted by delayed vascularization and fail to recapitulate this stepwise differentiation toward bone tissue. Here, we use the morphogen Sonic Hedgehog (Shh) to induce the in vitro organization of an endothelial capillary network in an artificial tissue. We show that endogenous Hedgehog activity regulates angiogenic genes and the formation of vascular lumens. Exogenous Shh further induces the in vitro development of the vasculature (vascular lumen formation, size, distribution). Upon implantation, the in vitro development of the vasculature improves the in vivo perfusion of the artificial tissue and is necessary to contribute to, and enhance, the formation of de novo mature bone tissue. Similar to the regenerating callus, the artificial tissue undergoes intramembranous and endochondral ossification and forms a trabecular-like bone organ including bone-marrow-like cavities. These findings open the door for new strategies to treat large bone defects by closely mimicking natural endochondral bone repair.

Engineered Micro-Objects as Scaffolding Elements in Cellular Building Blocks for Bottom-Up Tissue Engineering Approaches

A material-based bottom-up approach is proposed towards an assembly of cells and engineered micro-objects at the macroscale. We show how shape, size and wettability of engineered micro-objects play an important role in the behavior of cells on these objects. This approach can, among other applications, be used as a tool to engineer complex 3D tissues of clinically relevant size.

Fabrication of cell container arrays with overlaid surface topographies.

This paper presents cell culture substrates in the form of microcontainer arrays with overlaid surface topographies, and a technology for their fabrication. The new fabrication technology is based on microscale thermoforming of thin polymer films whose surfaces are topographically prepatterned on a micro- or nanoscale. For microthermoforming, we apply a new process on the basis of temporary back moulding of polymer films and use the novel concept of a perforated-sheet-like mould. Thermal micro- or nanoimprinting is applied for prepatterning. The novel cell container arrays are fabricated from polylactic acid (PLA) films. The thin-walled microcontainer structures have the shape of a spherical calotte merging into a hexagonal shape at their upper circumferential edges. In the arrays, the cell containers are arranged densely packed in honeycomb fashion. The inner surfaces of the highly curved container walls are provided with various topographical micro- and nanopatterns. For a first validation of the microcontainer arrays as in vitro cell culture substrates, C2C12 mouse premyoblasts are cultured in containers with microgrooved surfaces and shown to align along the grooves in the three-dimensional film substrates. In future stem-cell-biological and tissue engineering applications, microcontainers fabricated using the proposed technology may act as geometrically defined artificial microenvironments or niches.

Directed Assembly and Development of Material-Free Tissues with Complex Architectures

Material-free tissues are assembled using solely cells. Microstructured hydrogel templates and high content screening allow the formation of centimeter-scale tissues with precise architectures. Similar to developing tissues, these contract autonomously, controllably shift shape, self-scaffold by secreting extracellular matrix, and undergo morphogenesis.

Creating to understand – developmental biology meets engineering in Paris

In November 2016, developmental biologists, synthetic biologists and engineers gathered in Paris for a meeting called ‘Engineering the embryo’. The participants shared an interest in exploring how synthetic systems can reveal new principles of embryonic development, and how the in vitro manipulation and modeling of development using stem cells can be used to integrate ideas and expertise from physics, developmental biology and tissue engineering. As we review here, the conference pinpointed some of the challenges arising at the intersection of these fields, along with great enthusiasm for finding new approaches and collaborations. Summary: This Meeting Review summarises the recent ‘Engineering the embryo’ meeting, at which participants explored how synthetic systems can be used to understand principles of embryonic development.

Micro-aggregates do not influence bone marrow stromal cell chondrogenesis.

Although bone marrow stromal cells (BMSCs) appear promising for cartilage repair, current clinical results are suboptimal and the success of BMSC‐based therapies relies on a number of methodological improvements, among which is better understanding and control of their differentiation pathways. We investigated here the role of the cellular environment (paracrine vs juxtacrine signalling) in the chondrogenic differentiation of BMSCs. Bovine BMSCs were encapsulated in alginate beads, as dispersed cells or as small micro‐aggregates, to create different paracrine and juxtacrine signalling conditions. BMSCs were then cultured for 21 days with TGFβ3 added for 0, 7 or 21 days. Chondrogenic differentiation was assessed at the gene (type II and X collagens, aggrecan, TGFβ, sp7) and matrix (biochemical assays and histology) levels. The results showed that micro‐aggregates had no beneficial effects over dispersed cells: matrix production was similar, whereas chondrogenic marker gene expression was lower for the micro‐aggregates, under all TGFβ conditions tested. This weakened chondrogenic differentiation might be explained by a different cytoskeleton organization at day 0 in the micro‐aggregates. Copyright