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).

Human adipose stem/stromal cell (ASC) spheroids were used as a serum‐free in vitro model to recapitulate the molecular events and extracellular matrix organization that orchestrate a hypertrophic cartilage phenotype. Induced‐ASC spheroids (ø = 450 µm) showed high cell viability throughout the period of culture. The expression of collagen type X alpha 1 chain (COLXA1) and matrix metallopeptidase 13 (MMP‐13) was upregulated at week 2 in induced‐ASC spheroids compared with week 5 (P < .001) evaluated by quantitative real‐time PCR. In accordance, secreted levels of IL‐6 (P < .0001), IL‐8 (P < .0001), IL‐10 (P < .0001), bFGF (P < .001), VEGF (P < .0001), and RANTES (P < .0001) were the highest at week 2. Strong in situ staining for collagen type X and low staining for TSP‐1 was associated with the increase of hypertrophic genes expression at week 2 in induced‐ASC spheroids. Collagen type I, osteocalcin, biglycan, and tenascin C were detected at week 5 by in situ staining, in accordance with the highest expression of alkaline phosphatase (ALPL) gene and the presence of calcium deposits as evaluated by Alizarin Red O staining. Induced‐ASC spheroids showed a higher force required to compression at week 2 (P < .0001). The human ASC spheroids under serum‐free inducer medium and normoxic culture conditions were induced to a hypertrophic cartilage phenotype, opening a new perspective to recapitulate endochondral ossification in vivo.

A stabilized cartilage construct without signs of hypertrophy in chondrocytes is still a challenge. Suspensions of adipose stem/stromal cells (ASCs) and cartilage progenitor cells (CPCs) were seeded into micromolded nonadhesive hydrogel to produce spheroids (scaffold-and serum-free method) characterized by size, immunohistochemistry, fusion, and biomechanical properties. After cell dissociation, they were characterized for mesenchymal cell surface markers, cell viability, and quantitative real-time polymerase chain reaction. Both targeted and nontargeted (shotgun mass spectrometry) analyses were conducted on the culture supernatants. Induced ASC spheroids (ø = 350 mm) showed high cell viability and CD73 downregulation contrasting to CD90. The transforming growth factor (TGF)-b3/TGF-b1 ratio and SOX9 increased ( p < 0.05), whereas interleukin (IL)-6, IL-8, RUNX2, and ALPL decreased. Induced ASC spheroids were able to completely fuse and showed a higher force required to compression at day 14 ( p < 0.0001). Strong collagen type II in situ was associated with gradual decrease of collagen type X and a lower COLXA1 gene expression at day 14 compared with day 7 ( p = 0.0352). The comparison of the secretome content of induced and non-induced ASCs and CPCs identified 138 proteins directly relevant to chondrogenesis of 704 proteins in total. Although collagen X was absent, thrombospondin-1 (TSP-1), described as antiangiogenic and antihypertrophic, and cartilage oligomeric matrix protein (COMP), a biomarker of chondrogenesis, were upregulated in induced ASC spheroids. Our scaffoldand serum-free method mimics stable cartilage acting as a tool for biomarker discovery and for regenerative medicine protocols.

No abstract

Reproducibility is one of the basic assumptions in scientific experiments. If an experiment cannot be reproducible, its outcome cannot be proved and therefore, cannot be accepted [1]. This point has created great difficulties in biological field, for example, microbiology samples are strongly dependent on their strains, sometimes causing results inconsistency from different research groups using their own strains. In many cases there is mis identification or contamination of the samples. These difficulties are also observed in the field of Tissue Engineering.The expanding field of Tissue Engineering aims to build tissues and organs in vitro from three-dimensional cell culture systems suitable for regeneration, repair and / or replacement of damaged tissue. In order to achieve this goal, cells are cultivated in a threedimensional manner, using scaffold-dependent or scaffold-free strategies [2][3][4][5][6]. The first one is based on the combination of growth factors / genes with cells seeded in natural or synthetic biomaterials, which include ceramics, metal, polymers and decelularized matrices. These biomaterials should create a structural and molecular environment that mimics mechanical, geometrical and biological properties of the native organ in order to support the recipient´s cells [7-9]. The scaffold-free strategy is