A Layer-by-Layer Biofabrication for Bone Tissue Engineering

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1 A Layer-by-Layer Biofabrication for Bone Tissue Engineering GUDURIC Vera (INSERM U1026, Biotis, University of Bordeaux, Bordeaux, France) METZ Carole (INSERM U1026, Biotis, University of Bordeaux, Bordeaux, France) MAISANI Mathieu (Lab for Biomaterials and Bioengineering, CRC-I for the Innovation in Surgery, Quebec, Canada) SIADOUS Robin (INSERM U1026, Biotis, University of Bordeaux, Bordeaux, France) LEVATO Ricardo (Biomaterials for Regenerative Therapies Group, IBEC, Barcelona, Spain) BAREILLE Reine (INSERM U1026, Biotis, University of Bordeaux, Bordeaux, France) ENGEL Elisabeth (Biomaterials for Regenerative Therapies Group, IBEC, Barcelona, Spain) FRICAIN Jean-Christophe (INSERM U1026, Biotis, University of Bordeaux, Bordeaux, France) LUZANIN Ognjan (Faculty of Technical Sciences, University of Novi Sad, Novi Sad, Serbia) CATROS Sylvain (INSERM U1026, Biotis, University of Bordeaux, Bordeaux, France) Introduction The objectives of Bone Tissue Engineering (BTE) are to fabricate in vitro models of bone for cell biology studies and bone substitutes for regenerative medicine. Typical BTE approach requires cells specific to the target tissue, biochemical growth factors and a porous biocompatible scaffold as a support for cell proliferation and differentiation. Scaffolds for BTE are usually fabricated of natural biomaterials, such as agarose, alginate or collagen, but nevertheless of synthetic biomaterials, such as ceramic materials (ex. hydroxyapatite-ha), polyglycolic acid (PGA), polylactic acid (PLA), or hydrogels. They must possess specific features in terms of pore diameters, porosity and macroscopic dimensions. There are currently different methods to fabricate scaffolds for bone tissue engineering and rapid prototyping (RP) is of growing interest since it allows producing of custom tridimensional scaffolds with a high resolution. The conventional tissue engineering (TE) is based on seeding of macroporous scaffold on its surface ( down ᴀ Cells should populate and proliferate into the scaffold. The main approach is poor cell viability in the scaffold. Another approach, the approach, is based on assembly of small seeded blocks. Layer-by-Layer (LBL) microfabrication is based on this approach. The aim of this work was to evaluate cell 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 ᰀ戀漀琀琀漀

2 assembly of polylactic acid (PLA) membranes seeded with human stem cells. Materials and Methods PLA membranes were fabricated by direct 3D printing method which is an additive RP method based on extrusion of PLA dissolved in chloroform through a nozzle deposition process. The process was controlled using a tuned motor speed and pressure, in order to be adapted to viscosity of the solution. The motor speed was 3 mm/s and the pressure used was between psi, through a G27 nozzle. The membranes were 100 µm thick and their surface was 4 cm2. Two types of human primary cells were used for this work: HBMSCs isolated from femoral diaphysis bone marrow and EPCs isolated from cord blood. In order to be used for experiments, PLA membranes were cut with a tissue punch into 8 mm diameter circles. HBMSCs and EPCs were seeded onto membranes as mono(hbmscs cells/cm2, EPCs cells/cm2) and co-cultures HBMSCs /cm2, EPCs cells/cm2). Cell morphology (Scaning Electron Microscopy SEM), survival (Live-Dead essay), proliferation (DNA synthesis by CyQuant essay) and differentiation (alkaline phosphatase (ALP) for HBMSCs and Von Willebrand factor (VWF) for EPCs) were evaluated in 2D. Then, 3D LBL assemblies (4 layers) of the seeded membranes were prepared (one seeded membrane = one layer) either using fibrin glue either without it (Figure 1). Characterization of 3D constructs was performed using confocal microscopy and cryosesctions after specific phenotypic labeling of the cells (bottom-up approach). Control experiments were done by seeding cells after PLA membranes stacking (top-down approach). Results 2D mono- and co-cultures have shown cell survival on PLA during 21 days, using Live-Dead assay (Figure 2A). Proliferation in 2D displayed an increase of DNA synthesis in all cultures after 7 days (Figure 2B). Cell differentiation markers, ALP for HBMSCs and VWF for PECs, were expressed in all cultures. SEM showed the membrane topography and struts organization. It showed different cell morphology of the mono- and co-culture, as well as sample topography. Confocal microscopy (Figure 3) and histological analyses have shown viability and phenotype maintain of the cells implanted in the 3D LBL constructs (3 layers).

3 Discussion and Conclusion 2D evaluations of PLA seeded membranes gave promising results. Live-Dead assay showed survival of cells in all cultures during 21 days. Proliferation in 2D was increased in all conditions after 7 days of culture. Performing SEM, we could see different morphology of seeded cells. EPCs were small, rounded cells with flipodia towards PLA membranes. HBMSCs showed elongated and highly branched morphology. Co-cultures showed elongated and branched cells, as well as a large amount of extracellular matrix that covered the membrane pores. ALP expression was positive for both mono- and cocultures which evaluated early osteoblastic differentiation. Endothelial marker staining was maintained during 14 days. 3D experiments showed that LBL biofabrication enables better cell proliferation and differentiation into the scaffold than conventional BTE. The phenotype characterization of 3D constructs is ongoing. Different stabilization methods of 3D constructs could be used instead of fibrin glue and will be used in future experiments. Results obtained by now indicate that LBL approach could be suitable for bone tissue engineering, in order to promote homogenous cell distribution into the scaffold.

4 Figure 3. Confocal microscopy: osteoblastic phenotype characterized by CD90 (red), endothelial phenotype characterized by VWF (green) and nucleus label DAPI (blue)

5 Figure 2. A ጀ Live-Dead essay of PLA membranes seeded by mono- and co-cultures; B of mono- and co-cultures on PLA with control samples (cells without PLA) Figure 3. Confocal microscopy: osteoblastic phenotype characterized by CD90 (red), endothelial phenotype characterized by VWF (green) and nucleus label DAPI (blue) Acknowledgements Vera Guduric has obtained a French Government PhD Scholarship, issued by French Institute in Belgrade in Serbia. This scorarship is supported by Campus France (the French international agency for the promotion of higher education).