The hypertrophic cartilage induction influences the building-block capacity of human adipose stem/stromal cell spheroids for biofabrication.

As an alternative to the classical tissue engineering approach, bottom-up tissue engineering emerges using building blocks in bioassembly technologies. Spheroids can be used as building blocks to reach a highly complex ordered tissue by their fusion (bioassembly), representing the foundation of biofabrication. In this study, we analyzed the biomechanical properties and the fusion capacity of human adipose stem/stromal cell (ASC) we spheroids during an in vitro model of hypertrophic cartilage established by our research group. Hypertrophic induced-ASC spheroids showed a statistically significant higher Young's modulus at weeks 2 (P < .001) and 3 (P < .0005) compared with non-induced. After fusion, non-induced and induced-ASC spheroids increased the contact area and decreased their pairs' total length. At weeks 3 and 5, induced-ASC spheroids did not fuse completely, and the cells migrate preferentially in the fusion contact region. Alizarin red O staining showed the highest intensity of staining in the fused induced-ASC spheroids at week 5, together with intense staining for collagen type I and osteocalcin. Transmission electron microscopy and element content analysis (X-ray Energy Dispersive Spectroscopy) revealed in the fused quartet at week 3 a crystal-like structure. Hypertrophic induction interferes with the intrinsic capacity of spheroids to fuse. The measurements of contact between spheroids during the fusion process, together with the change in viscoelastic profile to the plastic, will impact the establishment of bioassembly protocols using hypertrophic induced-ASC spheroids as building blocks in biofabrication.

A Scaffold- and Serum-Free Method to Mimic Human Stable Cartilage Validated by Secretome.

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 µm) showed high cell viability and CD73 downregulation contrasting to CD90. The transforming growth factor (TGF)-ß3/TGF-ß1 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 scaffold- and serum-free method mimics stable cartilage acting as a tool for biomarker discovery and for regenerative medicine protocols. Impact Statement Promising adult stem cell sources for cartilage regeneration include adipose stem/stromal cells (ASCs) from subcutaneous adipose tissue. Our main objective was the development of a reproducible and easy-to-handle scaffold- and serum-free method to obtain stable cartilage from induced ASC spheroids. In addition to targeted protein profiling and biomechanical analysis, we provide the first characterization of the secretome composition for ASC spheroids, providing a useful tool to monitor in vitro chondrogenesis and a noninvasive quality control of tissue-engineered constructs. Furthermore, our secretome analysis revealed a potential novel biomarker-thrombospondin-1 (TSP-1), known by its antiangiogenic properties and recently described as an antihypertrophic protein.

Scaffold- and serum-free hypertrophic cartilage tissue engineering as an alternative approach for bone repair.

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.

Successful Low-Cost Scaffold-Free Cartilage Tissue Engineering Using Human Cartilage Progenitor Cell Spheroids Formed by Micromolded Nonadhesive Hydrogel.

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-ß1, TGF-ß2, and TGF-ß3 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).

Comparação da força resultante em barco "single skiff" na utilização de remos tipo "standard" e cutelo / Compare to the force applied by a rower to a boat

The aim of the study was to investigate the existence of a difference in the force appllied by a rower to a scull when two kinds of rowing blades are used (Macon blade, symnmetric and big blades, asymmetric) at the same stroke rate (number of rows per minute). The hypotheses of this study were that (1) there is a difference in the force applied by a rower to a boat when different kinds of rowing blades are used, and (2) the big blade will provide an increase in the force applied to move the bot compared to the Macon blade. A scull was tied to the four sides of a 25 m swimming pool by six steel cables: one on the bow, one on the stern, and four laterally in order to obtain the resultant force applied by the rower to the boat. Five rowers from three different rowing teams from Porto Alegre (Brazil) gave written consent to be the subjects of this study. All subjects rowed for five minutes with each blade, maintaining a stroke rate of approximately l0 strokes per minute. Root mean square (RMS) values were calculated for each force curve obtained. A t-test was used Lo compare the differences betwecn the RMS force values obtained (big blade B Macon blade) and a one-way ANOVA was used to verify if the group was heterogeneous. The level of significance adopted was 0.05. A 13.13 por cento difference was observed, indicating a higher efficiency of the big blade when compared to the Macon blade. The results presented in this study show that all athletes, using both blade types, were able to exert more force with the asymmeletric blade (big blade) than with the symmetric blade (Macon). These results suggest that the asymmetric blade provides a better performance at the same stroke rate during competition