Comparison of chondrogenic differentiation of mesenchymal stromal cells from human amniotic fluid and human adipose-derived tissue in chitosan-xanthan gum scaffolds

Introduction: After age and obesity, traumatic injuries represent the third most important risk factor for the development of osteoarthritis. Current treatments for cartilage injuries are not very effective. However, the use of stem cells, associated or not with scaffolds, has been proposed and investigated. In this study, we compared chondrogenic differentiation in human amniotic fluid mesenchymal stromal cells (hAF-MSC) and human adipose-derived mesenchymal stromal cells (hAD-MSC) grown in porous chitosan-xanthan gum scaffolds (CX) stimulated with TGF-β3, aiming at the possibility of direct implantation in the lesioned site. Methods: hAF-MSC were collected from women in the second trimester of pregnancy and hAD-MSC from patients that underwent liposuction. In the case of hAF-MSC samples, CD117-positive cells were selected. The mesenchymal stromal cells (MSCs) from both sources were expanded and characterized considering their capacity to adhere to polystyrene culture flasks, by flow cytometry analysis and differentiation into cartilage, bone and fat cells. The MSCs were seeded into chitosan-xanthan gum scaffolds specially designed for use in cartilage tissue engineering and grown under TGF-β3 stimulation. Differentiation was confirmed and evaluated by scanning electron microscopy (SEM), histology, immunohistochemistry and immunofluorescence analysis. Results: The results showed that MSCs from both sources exhibited high capacity for cell expansion, positivity for phenotypic markers, multipotency, chondrogenic potential and negativity for hematopoietic markers, in addition to differentiation capacity into the three above-mentioned mesenchymal lineages. Chondrogenic differentiation was confirmed by hematoxylin-eosin, alcian blue, picrosirius red and Masson's trichrome staining, indicating the presence of collagens and proteoglycans. Immunohistochemistry analysis showed positivity for collagen II and aggrecan, and immunofluorescence also showed positivity for collagen II. SEM revealed intense cell adhesion and collagen fibers adhered to the scaffold. Conclusions: In summary, it was possible to differentiate in vitro stem cells from human amniotic fluid and human adipose tissue into chondrocytes directly in the scaffold of chitosan and xanthan in the presence of TGF-β3, with evident production of an ECM rich in collagen and PGs.