Adipose Stromal Cell Spheroids for Cartilage Repair: A Promising Tool for Unveiling the Critical Maturation Point

Abstract

Articular cartilage lacks intrinsic regenerative capabilities, and the current treatments fail to regenerate damaged tissue and lead only to temporary pain relief. These limitations have prompted the development of tissue engineering approaches, including 3D culture systems. Thanks to their regenerative properties and capacity to recapitulate embryonic processes, spheroids obtained from mesenchymal stromal cells are increasingly studied as building blocks to obtain functional tissues. The aim of this study was to investigate the capacity of adipose stromal cells to assemble in spheroids and differentiate toward chondrogenic lineage from the perspective of cartilage repair. Spheroids were generated by two different methods (3D chips vs. Ultra-Low Attachment plates), differentiated towards chondrogenic lineage, and their properties were investigated using molecular biology analyses, biophysical measurement of mass density, weight, and size of spheroids, and confocal imaging. Overall, spheroids showed the ability to differentiate by expressing specific cartilaginous markers that correlate with their mass density, defining a critical point at which they start to mature. Considering the spheroid generation method, this pilot study suggested that spheroids obtained with chips are a promising tool for the generation of cartilage organoids that could be used for preclinical/clinical approaches, including personalized therapy.

Keywords: adipose stromal cells; chondrogenesis; deep imaging; mass density; spheroids.

Figure 1

Overall experimental procedure from ADSCs isolation and spheroids formation to molecular biology/biophysical parameters/deep imaging evaluations.

Figure 2

Violin plots showed gene expression at 7, 14, 21, and 28 days of the principal cartilaginous markers (A) Sox-9, (B) Collagen type II, and (C) Aggrecan. Data were normalized to GAPDH and analyzed using Student’s t-test for two-group comparisons with * p < 0.05. Different patterns were used for different spheroids culture methods: chips orange and ULA green.

Figure 3

(A) Representative images of spheroids cultured for 21 and 28 days on-chip or in ULA plates. The clarified spheroids were analyzed using confocal microscopy. The spheroids were stained with DAPI (blue) and anti-Collagen type II antibody (red). In the left panels, x-y single optical sections from the maximum diameter of the spheroids are shown, scale bars 50 µm. In the central panel, z-y cross sections are represented. The 3D rendering projections are focused on the right panels. (B) Spheroids volume after 21 or 28 days of differentiation on-chip or in ULA plates. Statistical analysis was performed using a two-tailed unpaired Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001. (C) Mean Fluorescence Intensity of anti-collagen type II antibody in spheroids cultured for 21 and 28 days on-chip or in ULA plates. The measurement is calculated in each optical section from the sum of the values of all the pixels divided by the number of voxels occupied by the spheroid. Statistical analysis was performed using two-tailed unpaired Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 4

On the top: violin plots showed measurement of mass density (A), weight (B), and diameter (C) of ADSCs spheroids cultured in ULA plates (green violins) or on-chip (orange violins) at 7, 14, 21, and 28 days of culture; on the bottom: statistical analysis performed using two-tailed unpaired Student’s t-test. The grid representation using colored squares corresponds as follows: white indicates no significance, yellow indicates p < 0.05, orange indicates p < 0.01, and red indicates p < 0.001.

Figure 5

Min-Max normalization of mass density ((A,D), depicted in violet), weight ((B,E), represented in blue), and diameter ((C,F), shown in grey) in relation to the gene expression levels of Sox-9 (shown in green), collagen type II (depicted in red), and aggrecan (displayed in yellow). The Min-Max normalization was computed over time for chips (A–C) and ULA (D–F). The values were expressed as a percentage of the data range.