Adult Stem Cells Spheroids to Optimize Cell Colonization in Scaffolds for Cartilage and Bone Tissue Engineering

Abstract

Top-down tissue engineering aims to produce functional tissues using biomaterials as scaffolds, thus providing cues for cell proliferation and differentiation. Conversely, the bottom-up approach aims to precondition cells to form modular tissues units (building-blocks) represented by spheroids. In spheroid culture, adult stem cells are responsible for their extracellular matrix synthesis, re-creating structures at the tissue level. Spheroids from adult stem cells can be considered as organoids, since stem cells recapitulate differentiation pathways and also represent a promising approach for identifying new molecular targets (biomarkers) for diagnosis and therapy. Currently, spheroids can be used for scaffold-free (developmental engineering) or scaffold-based approaches. The scaffold promotes better spatial organization of individual spheroids and provides a defined geometry for their 3D assembly in larger and complex tissues. Furthermore, spheroids exhibit potent angiogenic and vasculogenic capacity and serve as efficient vascularization units in porous scaffolds for bone tissue engineering. An automated combinatorial approach that integrates spheroids into scaffolds is starting to be investigated for macro-scale tissue biofabrication.

Keywords: adult stem cells; biofabrication; building-blocks; scaffolds; spheroids.

Figure 1

Association of ASC spheroids induced for chondrogenic pathway in PCL nanofibers. (A) Aligned PCL nanofibers, (B) induced ASC spheroids associated with PCL nanofibers, (C) initial stage of induced ASC spheroids fusion, (D) induced ASC spheroids associated with PCL nanofibers incubated with cytoplasmic calcein (green staining) and ethidium homodimer (red staining) revealed mostly viable cells and few necrotic cells (arrow). (E,F) scanning electron microscopy of induced ASC spheroids associated with PCL nanofibers showing initial stages of spheroids proximity (E) and fusion (F).

Figure 2

Scanning electron microscope of ASC spheroids associated with 3D PLA/CHA printed scaffold. (A) Two spheroids in different areas of scaffold. Bar size: 200 μm; (B) Cells of spheroid interacting with the biomaterial surface. Bar size: 50 μm; (C) Note the presence of filopodia (arrow) produced by spheroid cells promoting a better interaction with the scaffold. Bar size: 20 μm.

Figure 3

Automated platform for spheroids seeding into 3D printed scaffolds. (A) The bioprinter; (B) spheroids are dispensed by the bioprinter into the 3D printed scaffolds; (C) the 3D printed scaffold; (D) the bioprinter dispensing one layer of spheroids for each layer of the 3D printed scaffold. The state-of-art is one spheroid seeded in each spacing of the 3D printed scaffold.