B. Barzaghini

C. Cereda

E. Jacchetti

L. Messa

M. T. Raimondi

S. Carelli

T. Baldissera

English

Archivio istituzionale della ricerca - Politecnico di Milano

XII Annual Meeting of the Italian Chapter of the European Society of Biomechanics September 18-19, 2023, Turin, Italy 2        5 XII Annual Meeting of the Italian Chapter of the European Society of Biomechanics September 18-19, 2023, Turin, Italy 36  3D MICROSTRUCTURED SCAFFOLD GEOMETRY DRIVES MESENCHIMAL STEM CELL PHENOTYPE  Emanuela Jacchetti (1), Teresa Baldissera (1), Bianca Barzaghini (1), Letizia Messa (2,3), Cristina Cereda (4), Stephana Carelli (2,4), Manuela Teresa Raimondi (1) 1. Department of Chemistry, Materials and Chemical Engineering, Politecnico di Milano, Italy. 2. Pediatric Research Center "Romeo ed Enrica Invernizzi, University of Milano, Milano, Italy. 3. Department of Electronics, Information and Bioengineering, Politecnico di Milano, Italy. 4. Center of Functional Genomics and Rare Diseases, Buzzi Childen's Hospital, Milano, Italy.  Introduction Mechanical stimuli from the environment affect mesenchymal stem cells (MSCs) morphology and functionality [1]. We observed an increased stemness maintenance for MSCs grown into a 3D custom-made scaffold [2]. We are now investigating the role of scaffold architecture in maximizing the cell stemness maintenance over the time, and the identification of molecular pathways most significantly deregulated. Methods Custom made organic-inorganic polymeric scaffolds were produced by the two-photon polymerization technique with different dimension of cubic pores (15x15x15 m3, 20x20x20 m3) or graded pore (Named Nichoid), with a range between 10 and 30 μm transversely and 15 μm in the vertical direction.  Fig.1: Representative portion of the 3D investigated scaffolds (top view). Finite element analyses (COMSOL Multiphysics®) were performed to evaluate the displacement (di) and bending and longitudinal stiffnesses (Ki=Fi/di) of each pores type. We built the model in stationary condition, and linear elastic and isotropic material properties (E=3.03GPa, =0.49, =1200kg/m3) were imposed. We tested three load cases (|F|=70nN), one for each direction, placed at half-length of bars for representative pores (Fig.1).  Fig.2: Example of a meshed scaffold composed by graded pores, meshed by COMSOL Multiphysics®.  104MSCs were cultured for 24h and 7days, in 3D and 2D samples, using standard culture conditions. As pharmacological control, 1 m Cytochalasin-D for 1h was used to reduce internal cellular tension. Fluorescence imaging was performed to investigate cell migration capability in situ and ex situ, nuclear morphology and nucleoskeletal organization. RNA-Seq and Bioinformatic Data Analysis were used toinvestigate the gene expression deregulation induced by the different scaffolds. Results RNA-Seq analyses indicate that culture conditions significantly affect gene expression. Among all the 3D conditions tested, cell stemness significantly increased only in Nichoid scaffolds. Computational analysis demonstrates that this condition induces increased gradients in the displacement and stiffness of the microscopic trusses that form the pore microgrid (Fig.2a,b). Fluorescence imaging shows that the 3D scaffold geometry does not affect the organization of several proteins primarily involved in the mechanotransduction pathway; on the contrary, it influences significantly the cell capability to migrate (Fig.2c).  Fig.2: a) Representation of truss displacement under the point load Fx=70nN. b) Pore stiffness along the three investigated directions; c) Analysis of MSCs capability to migrate. Discussion MSCs cultured in our 3D scaffolds do not show a significant cell reshaping and remodelling of the main structural proteins; however, it is appreciable a significative gene deregulation. Our hypothesis is that this phenomenon is guided by a modulation of the cell confinement and migration properties. Our findings reveal novel aspects of the MSCs culture in 3D, representing a step forward in the control of stem cells via purely mechanical conditioning, thus paving the way to new strategies for MSCs translation to clinical applications. References 1. Steward AJ et al. J Anat. 2015;227(6):717-31. 2. Remuzzi A. et al. Cells 2020, 9, 1873 Acknowledgements ERC, project BEACONSANDEGG, G.A. 101053122; EU, FET-OPEN project IN2SIGHT, G.A. 964481; NICHILD - 101068512 - GAP-101068512 

3D microstructured scaffold geometry drives mesenchimal stem cell phenotype

https://re.public.polimi.it/bitstream/11311/1286522/1/Jacchetti_ESBITA_2023.pdf

3D microstructured scaffold geometry drives mesenchimal stem cell phenotype

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Archivio istituzionale della ricerca - Politecnico di Milano