Microscopic evidence of the primary astrocytes' morphological differentiation and migration inside porous Poly-L-lactic acid 3D‑scaffolds

Abstract

Tissue engineering is an emerging multidisciplinary field that aims at reproducing in vitro and/or in vivo tissues with morphological and functional features similar to the biological tissue of the human body [1]. In the attempt to construct suitable tissue models, a critical step is the setting of 3D scaffolds that mimic the supportive structures of a natural extracellular matrix microenvironment into which cells are normally embedded. In this context, the generation of 3D cultures of brain cells is of particular interest. For instance, the poly L‐lactic acid (PLLA) polymer is wildly used because of its biocompatible and biodegradable potential; the PLLA scaffold topography simulates the natural extracellular matrix (ECM) and can make it a good candidate for neural tissue engineering [2]. To achieve this goal, in this study, PLLA scaffold with characteristics of bioactivity was prepared via thermally-induced phase separation (TIPS) [3], and utilized as substrate for primary rat astrocytes 3D growth. To assess the cells spatial distribution and morphology within the scaffolds, the structures were characterized by scanning electron microscopy. For comparison, astrocytes were also cultured in the traditional 2D culture system that we have been using since 2003. Different scaffold morphologies and coatings such as collagen I and IV, and fibronectin were tested in order to evaluate their influence on astrocyte growth, morphology and EV production. To evaluate these effects on astrocyte morphology on the PLLA scaffolds, TEM preparation was also performed. Cells were present in all regions of the scaffold, they were observed to adhere, grow and penetrating into the interior region of the scaffold, acquiring their typical morphology. In addition, they also secrete EVs as in vivo [4]. Their ability to produce EVs was demonstrated by both TEM and SEM analyses, which revealed intracellular MVBs and EVs compatible in size with exosomes. The results revealed that the porous sheath of PLLA allowed cell migration inside the scaffold and that the one coated with collagen IV, served as very good matrices for astrocytes, suggesting that the chosen conditions could be a good starting point for the preparation of 3D brain cell co culture systems useful for clinical applications. References [1] R. Langer, JP. Vacanti. Science. 14 260 (1993): 920-926 [2] M. Soleimani, S. Nadri, I. Shabani. Int. J. Dev. Biol. 54 (2010) 1295-1300 [3] GA. Mannella, G. Conoscenti, F. Carfì Pavia, V. La Carrubba, V. Brucato. Mater Lett 160 (2015) 31-33 [4] G. Schiera, CM. Di Liegro, I. Di Liegro. Biomed Res Int (2015): 152926