In native bone, osteoblasts (bone cells), osteogenic precursors and endothelial cells (cells that line the interior surface of blood vessels) interact in a synergetic way towards the coordinated development of vasculature and mineralized tissue; therefore, close spatial relationships are established between the two tissues in the forming bone: the vascular network acts as a ‘template’ for the deposition of bone mineral [1] (Figure 1). The sheer complexity of this collaborative assembly/growth process, increased by the difficulty of studying it in vivo, results in a limited understanding of the mechanisms guiding interactions between endothelial cells and osteogenic precursors. This explains why tissue-engineered vascularized bone grafts continue to represent a technical and scientific challenge. Indeed, despite promising and exciting results, the progression of this field of medicine is still hampered by challenging bottlenecks such as selecting most effective cell types, designing mechanically-compatible porous scaffolds, combining efficient growth factors, selecting proper immunomodulatory biomaterials or agents, achieving a proper vascularization, and most importantly controlling quality and function of the regenerated bone.

Angiogenesis, the formation of new blood vessels by a process of sprouting from pre-existing ones, is also critical for the establishment and maintenance of large engineered tissues, and as known, vascularization is a critical challenge in tissue engineering. Here, an important target is to develop proper model systems from 2D to 3D, mimicking more complex environment of 3D scaffolds, allowing to simulate the formation of blood vessels in a reconstructing bone, starting from colonization by endothelial cells forming first a closed-up channel, followed by vessel stabilization by pericytes or smooth muscle cells (cells that allow the vessel stabilization). These model systems should have important characteristics such as the possibility to integrate different cells in order to mimic the different cellular layers of real blood vessels; the possibility to have cells submitted to a continuous flow as in blood vessels; and the possibility to deliver signaling cues by grafting or embedding biological factors.

The goals of the present PhD project may be summarized as follows :
 - microfabricate porous channels in bio-relevant materials as model systems for channels in real 3D scaffolds.
- functionalize the channels with bioactive active principles or incubate cells with active vesicles able to favour vascularization
 - develop a model system for the study of vascularization in conditions allowing to control softness, biochemistry and environmental parameters (flow).

Research Methodology

In view of these challenges, this PhD project is planned, pooling the expertise of researchers in materials science and biology of 2 laboratories and 1 private sector partner:
-    Institute of Chemistry & Biology of Membranes & Nanoobjects (CBMN) Bordeaux
-    Institute of Condensed Matter and Nanosciences (IMCN) Louvain la Neuve 
-    It4ip (Louvain-la-Neuve, Belgium)

This thesis  (UBordeaux-UCLouvain) will be centered on the development of closed microchannels of functional biocompatible materials designed for the enhancement of vascular reconstruction, with functionalization of the channel walls, and with the possibility to embed the system in a microfluidic system for flow stimulation. The thesis will be directed jointly by Drs. MC. Durrieu and L. Plawinski as experts in bone and vascular tissue engineering and bioactive surface functionalization and Profs. S. Demoustier-Champagne and A.M. Jonas as material scientists specialized in surface (bio)functionalization and materials micro/nano-shaping and processing . Two different types of materials will be explored for the channels, soft crosslinked gels of biopolymers and hard channels made of polycarbonate. In the latter case, the material will be perforated by nanopores at specific locations for cell feeding, using track etching technologies developed by the associated industrial partner it4ip (Figure 2).

Figure 2: Example of nanopores created by ion-track etching technologies in a polymer thin film. Similar pores will be made in polymer films with pre-molded grooves.

More specifically, open microchannels will be created by micromolding, embossing or standard photolithography by the UCLouvain partner in either crosslinked gels of biopolymers such as hyaluronic acid, chitosan, poly(glutamic acid) or denatured collagen, or in polycarbonate. For polycarbonate, processing of these systems in close collaboration with the it4ip associated industrial partner, will result in the formation of pores at specific locations along the channels. The channels will differ in softness of their material (soft gel or hard polycarbonate), size (depth and width), tortuosity or surface functionality (post-functionalization will be performed by UBordeaux and UCLouvain, using mainly bioactive peptides); arrays of criss-crossed channels might also be explored if time permits. Cell culture (Human Umbilical Vein Endothelial Cells (HUVECs) purchased from PromoCell) will be performed in static conditions by the UBordeaux partner: Dr MC Durrieu previously developed 2D bioactive functionalized materials (polymer surfaces functionalized with SVVYGLR peptides) able to induce endothelial cell morphogenesis into tube formation . Previous studies performed by Dr L. Plawinski showed that endothelial microvesicles introduced in culture medium modulate the angiogenic responses of HUVECs . We propose two strategies to induce angiogenesis: to graft SVVYGLR peptides onto the microchannel wall or to incubate cells with endothelial microvesicles. In addition, possible inclusion of the microchannels into microfluidic setups will be considered. At the end of the thesis, a range of fabrication methodologies for functional microchannels with possible nanoporosity will be available; in addition, knowledge of the main parameters controlling angiogenesis in such model systems will have been acquired.

[1] Correia et al., PLoS one 2011, 6, e28352

Project Partners

Institute of Chemistry & Biology of Membranes & Nanoobjects, Univesity Bordeaux, France

Institute of Condensed Matter and Nanosciences, Université catholique de Louvain, Belgium

Industry Partner: it4ip, Belgium

Host Country (employment): France

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