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.

The scaffold-free tissue engineering using spheroids is pointed out as an approach for optimizing the delivery system of cartilage construct. In this study, we aimed to evaluate the micromolded nonadhesive hydrogel (MicroTissues®) for spheroid compaction (2-day culture) and spontaneous chondrogenesis (21-day culture) using cartilage progenitors cells (CPCs) from human nasal septum without chondrogenic stimulus. CPC spheroids showed diameter stability (486 μm ± 65), high percentage of viable cells (88.1 ± 2.1), and low percentage of apoptotic cells (2.3%). After spheroid compaction, the synthesis of TGF-β1, TGF-β2, and TGF-β3 was significantly higher compared to monolayer (p < 0.005). Biomechanical assay revealed that the maximum forces applied to spheroids after chondrogenesis were 2.6 times higher than for those cultured for 2 days. After spontaneous chondrogenesis, CPC spheroids were entirely positive for N-cadherin, collagen type II and type VI, and aggrecan and chondroitin sulfate. Comparing to monolayer, the expression of SOX5 and SOX6 genes analyzed by qPCR was significantly upregulated (p < 0.01). Finally, we observed the capacity of CPC spheroids starting to fuse. To the best of our knowledge, this is the first time in the scientific literature that human CPC spheroids were formed by micromolded nonadhesive hydrogel, achieving a successful scaffold-free cartilage engineering without chondrogenic stimulus (low cost).

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