Comparision between human amniotic fluid and adipous tissue mesenchymal stem cells induced-chondrogenesis cultured in chitosan-xanthan scaffold stimulated with TGF-β3

Abstract

Purpose: The use of stem cells (SC) became a promising therapy for regenerative medicine due to their high plasticity and pluripotency. Such characteristics could be useful for the development of effective treatments not only for cartilage pathologies such as the ones resulting from trauma, but also for diseases, like osteoarthritis (OA). The aim of this study was to compare human amniotic fluid (HAF) mesenchymal stem cells induced chondrogenesis, with adipose tissue (AT) cells induced chondrogenesis in a chitosan-xanthan scaffold, both stimulated with TGF-beta 3; also to evaluate the influence of this scaffold in the differentiation process of both cells lineage. Methods: HAF were obtained from amniocentesis of 43 pregnant women in the 2nd trimester, who agreed to participate after sign a free and informed consent form (the study was approved by the local research ethical board). After separation the SCs were cultured in growth medium, expanded and selected by immunomagnetic separation for those CD117+ (cKit). AT were obtained from patients who underwent to liposuction in plastic surgery unit. The SCs from AT were isolated, cultured and expanded. The Scs from both sources were characterized, expanded up to four passages and from that stage, submitted to the same procedures as follow: inserted into the scaffold at the concentration of 2x106 in each one and cultured in chondrogenic medium supplemented with TGF-beta3, 10 ng/ml for 21 days. The chondrogenic differentiation was qualitatively analyzed by histology with Hematoxylin-Eosin (HE), Masson's Trichrome (MT), Picrossirius Red (PiRed) and Alcian Blue (AB); immune. Through SEM, intense cellularhistochemistry and Scanning Electron Microscopy (SEM) were also used in the characterization. Results: The SC from both sources remained stable in culture, adhered on the flasks as fibroblasts-like forming colony. The Flow Cytometry from both sources was positive for characterisc MSC markers, high positivity for markers expressed in cells condensation, indicating high chondrogenic potential and negativity for hematogenic lineage markers. It was also observed the SC ability, from both studied sources, to differentiate in the three mesenchymal lineages, adipogenic, osteogenic and chondrogenic. After characterization and cells insertion into the scaffolds, the construct was cultured for 21 days. The histologic analysis showed the affinity of cells to the scaffold, a large extracellular matrix production, rich in collagen and proteoglycans and the efficacy of TGF-beta3. The presence of collagen type II and aggrecan was demonstrated by immunehistochemistry positivity for them. Through SEM, intense cellular growth and collagen fibers production, strongly adhered to the scaffold were observed. Conclusions: The HAF SCs presented greater adhesion to the scaffold and resistance to the steps of the study than AT SC. Therefore we concluded that although both stem cells sources were feasible for application in chondrogenic experiments, as far as the chondrogenic differentiation into the scaffold of chitoman-xanthan under TGF-beta 3 stimuli, the HAF SC seems to be , at least qualitatively, better than AT SC.