Amniotic fluid MSCs for scaffold-free cartilage repair: spheroid fusion and chondrogenic microtissue development

Abstract

Background: Articular cartilage injuries are challenging due to limited regenerative capacity, causing chronic pain and impaired mobility. Current treatments are often inadequate, necessitating novel cartilage repair approaches. This study investigates amniotic fluid-derived mesenchymal stromal cells (AF-MSC) as a promising cell source for tissue engineering.

Research design and method: Cartilage-like microtissues were produced by differentiating AF-MSC into chondrocytes within a 3D culture system. Using a 3D-printed non-adhesive micromold, AF-MSC spheroids were formed and fused into larger microtissues. Spheroids were characterized for morphology, viability, and extracellular matrix (ECM) production. The mechanical properties of resulting microtissues were compared to native cartilage and agarose hydrogel.

Results: AF-MSC proved a viable, scalable cell source for cartilage microtissues. Spheroid fusion created structures with mechanical properties and ECM components resembling native cartilage.

Conclusions: AF-MSCs differentiated into chondrocytes when stimulated with TGF-β3 in a 3D micromolded culture, forming uniform, viable spheroids with robust ECM production and mechanical properties. These spheroids fused into neocartilage microtissue, showing potential for regenerative medicine, especially osteoarthritis treatment and drug testing. Further research should optimize conditions and evaluate long-term biomechanical performance.

Keywords: Mesenchymal stem cells; amniotic fluid; chondrogenesis; tissue engineering and cartilage regeneration.

Graphical abstract

Figure 1.

Morphological characterization, differentiation test, and immunophenotyping of AF-MSC. Representative image of human amniotic fluid cells in cellular expansion exhibiting fibroblast-like colony-forming morphology. Phase contrast microscope (400X) (A); Cells differentiated into adipocytes with fat vesicles stained orange by Oil Red (B); osteocytes with calcium crystals stained red by Alizarin Red (C); chondrocytes with glycosaminoglycan production stained blue by Alcian Blue (D); Immunophenotypic analysis by flow cytometry of mesenchymal markers (E), pluripotency markers (F), and indicators of higher chondrogenic potential markers (G). Histograms represent the fluorescence intensity of each marker by the number of counted events.

Figure 2.

Characterization of produced spheroids regarding morphology, viability, and spontaneous fusion capacity. SEM images of a uniform group of spheroids (A); morphology of a single spheroid (B); internal porous structure of a spheroid in cross-section (C); detailed surface view showing a compact extracellular matrix (ECM) with a high number of cells (arrows) (D). Viability over the cultivation period. Viable cells were stained green, and dead cells were stained red using the LIVE/DEAD technique (E, F, G, H). Images were obtained using a confocal microscope. Fusion dynamics. Spheroids in the micromold (I); spheroids grouped in a single non-adhesive well after 24 hours (J); completely fused spheroids after 72 hours. Images were obtained using an inverted optical microscope (40X magnification).

Figure 3.

Microtissue production through sequential spheroid fusion: macroscopic and histological analysis. The images depict the structural evolution during the culture process, with histological staining at different stages. Hematoxylin and eosin staining highlights cell nuclei (purple) and extracellular matrix (pink) (A–C). Alcian Blue staining indicates glycosaminoglycans (light blue) (D–F), while Masson’s trichrome reveals collagen fibers (blue) (G–I). Picrosirius Red staining further highlights collagen fibers (red) throughout the tissue (J–O). Black arrows mark spheroid adhesion regions (M), with cellular migration contributing to fusion (N) and extensive extracellular matrix deposition (O).

Figure 4.

Quantification of matrix components using immunostaining. Semi-quantitative analysis of immunohistochemical data for collagen type II and aggrecan in each group, represented as a percentage of the labeled area using ImageJ software, is presented in the graph. (A) In the immunohistochemical staining, positive labeling for type II collagen antibodies (B, C, D) and aggrecan (E, F, G) is shown in brown, while nuclei were counterstained in purple with hematoxylin. A negative control was included to assess immunoreactivity (H, I, J). The antibodies used were: Rabbit Polyclonal Anti-Collagen II Antibody, BIOSS, and Rabbit Polyclonal Anti-Aggrecan Antibody, BIOSS.

Figure 5.

Mechanical Properties of Cartilaginous Microtissue. Comparative analysis between the microtissue and human knee cartilage samples (A). The shape and dimensions of each sample are described (B), and the mechanical properties are shown (C). The compressive behavior of each material is represented in the graph during testing. Samples were subjected to compressive loading until 100% deformation, at which point the maximum compressive stress (MCS) was recorded (D).