| المؤلفون: |
Côrtes I; Laboratory of Tissue Bioengineering, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.; Post-Graduation Program of Translational Biomedicine (Biotrans), Unigranrio, Campus I, Duque de Caxias, Brazil.; Multidisciplinary Center for Biological Research (Numpex-Bio), Federal University of Rio de Janeiro (UFRJ) Xerém, Duque de Caxias, Brazil., Matsui RAM; Laboratory of Tissue Bioengineering, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.; Post-Graduation Program of Translational Biomedicine (Biotrans), Unigranrio, Campus I, Duque de Caxias, Brazil.; Multidisciplinary Center for Biological Research (Numpex-Bio), Federal University of Rio de Janeiro (UFRJ) Xerém, Duque de Caxias, Brazil., Azevedo MS; Multidisciplinary Center for Biological Research (Numpex-Bio), Federal University of Rio de Janeiro (UFRJ) Xerém, Duque de Caxias, Brazil., Beatrici A; Scientific and Technological Metrology Division (Dimci), National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.; Post-Graduation Program in Biotechnology, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil., Souza KLA; Multidisciplinary Center for Biological Research (Numpex-Bio), Federal University of Rio de Janeiro (UFRJ) Xerém, Duque de Caxias, Brazil., Launay G; Molecular Microbiology and Structural Biochemistry, UMR 5086, University of Lyon, CNRS, Lyon, France., Delolme F; Tissue Biology and Therapeutic Engineering Laboratory, UMR 5305, University of Lyon, CNRS, Lyon, France.; SFR Biosciences, ENS de Lyon, INSERM US8, CNRS UMS3444, University of Lyon, Lyon, France., Granjeiro JM; Laboratory of Tissue Bioengineering, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.; Post-Graduation Program of Translational Biomedicine (Biotrans), Unigranrio, Campus I, Duque de Caxias, Brazil.; Post-Graduation Program in Biotechnology, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.; Laboratory of Clinical Research in Odontology, Fluminense Federal University (UFF), Niterói, Brazil., Moali C; Tissue Biology and Therapeutic Engineering Laboratory, UMR 5305, University of Lyon, CNRS, Lyon, France., Baptista LS; Laboratory of Tissue Bioengineering, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.; Post-Graduation Program of Translational Biomedicine (Biotrans), Unigranrio, Campus I, Duque de Caxias, Brazil.; Multidisciplinary Center for Biological Research (Numpex-Bio), Federal University of Rio de Janeiro (UFRJ) Xerém, Duque de Caxias, Brazil.; Post-Graduation Program in Biotechnology, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil. |
| مستخلص: |
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)-β3/TGF-β1 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. |
