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Título/Title/Titulo

NON-HYPERTROPHIC CARTILAGE BY SCAFFOLD-FREE TISSUE ENGINEERING VALIDATED BY COMPARATIVE SECRETOME ANALYSIS

Introdução/Introduction/Introdución

Articular cartilage has a poor capacity of regeneration and promising adult stem cell sources for cartilage regeneration include adipose stem/stromal cells (ASCs) from subcutaneous adipose tissue.

Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados

In this study, we attempted to engineer a non-hypertrophic cartilage scaffold-free tissue engineering, represented by spheroids. Human lipoaspirate samples and cartilage fragments from nasal septum were obtained from healthy donors that underwent aesthetic surgery procedures (Protocols 145-09 and 146-09). Cell suspension was seeded in a micromolded agarose hydrogel for spheroids formation for three experimental groups: Induced ASC spheroids; Non-induced ASC spheroids and Cartilage progenitor cells (CPC) spheroids. For chondrogenic induction, ASC spheroids were maintained under hypoxia (10% O2 and 5% CO2) in culture medium supplemented with 10 ng/ml TGF-β3 and 10-8 M dexamethasone. ASC and CPC spheroids were kept at 37 °C with 5% CO2 and atmospheric O2, in absence of chondrogenic inducers. All experimental groups were maintained for until 21 days. Induced and non-induced ASC spheroids showed most viable cells identified by 7AAD exclusion strategy. The level of TGF-β1 in supernatant evaluated by multiplex assay of induced ASC spheroids significantly decreases comparing day 21 to 7 (p = 0.0020) and low level of TGF-β3 was detected in supernatant of non-induced ASC spheroids at days 7 (p = 0.0022), 14 (p = 0.0022) and 21 (p = 0.0022) compared to induced ASC spheroids. Related to interleukins levels, the supernatant of non-induced and induced ASC spheroids showed similar high levels for IL-6 (p = 0.3282) and IL-8 (p = 0.7137) at day 7. The supernatant of induced ASC spheroids showed a decrease at day 21 for IL-6 (p < 0.0001) and for IL-8 (p < 0.0001) compared to day 7. The master gene for chondrogenesis – SOX9 – was significantly upregulated at day 14 (p = 0.0436) and day 21 (p = 0.0004) compared to day 7 in induced ASC spheroids. SOX5 (p = 0.0029) and SOX6 (p < 0.0001) were significantly upregulated at day 21 compared to day 7. Induced ASC spheroids were completely positive for collagen type I, type II, type VI and aggrecan. The intensity of staining for collagen type I was low compared to collagen type II, the typical collagen from hyaline cartilage. Interestingly, collagen type X, a typical collagen from hypertrophic cartilage, decreased its intensity along induced ASC spheroids culture. Induced ASC spheroids showed a significantly high force module at day 7 (p = 0.0003), 14 (p < 0.0001) and 21 (p = 0.0007) compared to non-induced ASC spheroids evaluated by Microsquiser equipment. Secretome (proteomic of cell culture supernatant at day 21) was analyzed by LC-MS/MS. Experiments were performed on a QExactive HF mass spectrometer operating with Xcalibur software (version 4.0). We identified up to 644 proteins, including proteins related to skeletal development, extracellular matrix remodeling and collagen synthesis.

Considerações Finais/Final considerations/Consideraciones finales

ASC induced spheroids showed high viability, a high level of TGF- β3 and SOX-9 gene expression besides a high force module. These spheroids showed the typical extracellular matrix components of cartilage, most of them confirmed in secretome. More importantly the collagen type X was not identified in secretome and its intensity of staining decreases along induced ASC spheroid culture. We can conclude that a non-hypertrophic phenotype was reached by induced ASC spheroids.

Palavras-chave/Key words/Palabras clave

Secretome, Spheroids, Adipose derived Stem/Stromal Cells (ASCs), chondrogenesis.