Successful Low-Cost Scaffold-Free Cartilage Tissue Engineering Using Human Cartilage Progenitor Cell Spheroids Formed by Micromolded Nonadhesive Hydrogel

Abstract

The scaffold-free tissue engineering using spheroids is pointed out as an approach for optimizing the delivery system of cartilage construct. In this study, we aimed to evaluate the micromolded nonadhesive hydrogel (MicroTissues®) for spheroid compaction (2-day culture) and spontaneous chondrogenesis (21-day culture) using cartilage progenitors cells (CPCs) from human nasal septum without chondrogenic stimulus. CPC spheroids showed diameter stability (486 μm ± 65), high percentage of viable cells (88.1 ± 2.1), and low percentage of apoptotic cells (2.3%). After spheroid compaction, the synthesis of TGF-β1, TGF-β2, and TGF-β3 was significantly higher compared to monolayer (p < 0.005). Biomechanical assay revealed that the maximum forces applied to spheroids after chondrogenesis were 2.6 times higher than for those cultured for 2 days. After spontaneous chondrogenesis, CPC spheroids were entirely positive for N-cadherin, collagen type II and type VI, and aggrecan and chondroitin sulfate. Comparing to monolayer, the expression of SOX5 and SOX6 genes analyzed by qPCR was significantly upregulated (p < 0.01). Finally, we observed the capacity of CPC spheroids starting to fuse. To the best of our knowledge, this is the first time in the scientific literature that human CPC spheroids were formed by micromolded nonadhesive hydrogel, achieving a successful scaffold-free cartilage engineering without chondrogenic stimulus (low cost).

Figure 1

Scheme of cartilage progenitor cell spheroid culture. (a) Silicone molds were used from MicroTissues 3D Petri Dish. (b) The agarose solution was dispensed into silicone molds, resulting in the micromolded nonadhesive agarose hydrogel (c). (d) The cell suspension was carefully seeded into cell seeding chamber with single pipetting. (e) The cell suspension decanted into circular recesses after few minutes resulting in spheroids after 18 hours (f).

Figure 2

The CPC spheroids formed in the micromolded nonadhesive hydrogel with 800 μm diameter in each circular recesses showed stability in their diameter along 21 days of cell spheroid culture. (a, c) Representative images of CPC spheroids formed in micromolded nonadhesive hydrogel with 300 μm diameter (a) and with 800 μm diameter in each circular recesses (c). Phase contrast microscopy. Bar size: 100 μm. (b, d) The graphs represent the mean ± standard error of the spheroid diameter in micromolded nonadhesive hydrogel with 300 μm diameter (b) and with 800 μm diameter in each circular recesses (d). Light gray bar represents 2 days of cell spheroid culture. Dark gray bar represents 21 days of cell spheroid culture. ^∗∗∗∗p < 0.0001; ^∗∗p < 0.01.

Figure 3

The CPC spheroids formed in the micromolded nonadhesive hydrogel with 800 μm diameter in each circular recesses showed a high percentage of viability after spontaneous chondrogenesis. (a, b) Dot-plot graphs showing CPC positive for only CD44 in blue and double positive for CD44 and 7AAD in pink, after digestion of spheroids cultivated for 2 (a) and 21 (b) days. The viable cells were identified by 7AAD exclusion, outside the region R1. (c) The graph represents the percentage of viable cells in three different samples (n = 3). (d, e) Dot-plot graphs of annexin V and PI evaluation showing CPC positive for annexin V in Q2 and Q4, after digestion of spheroids cultivated for 2 (d) and 21 (e) days. (f) The graph represents the percentage of apoptotic cells (annexin V positive cells) in three different samples (n = 3). 7AAD: 7-actinomycin D; CD: cell cluster of differentiation; PI: propidium iodide.

Figure 4

The CPC spheroids formed in the micromolded nonadhesive hydrogel with 800 μm diameter in each circular recesses showed an increase in TGF-β1, TGF-β2, and TGF-β3 synthesis comparing to CPCs monolayer. The CPC spheroids maintained for 2 days (spheroid compaction) in cell spheroid culture showed the highest synthesis of TGF-β1 (a), TGF-β2 (b), and TGF-β3 (c) comparing to the CPC spheroids maintained for 21 days (spontaneous chondrogenesis) and to monolayer (^∗∗p < 0.01, ^∗∗∗p < 0.005). TGF-beta: transforming growth factor-beta.

Figure 5

The CPC spheroids formed in the hydrogel with 800 μm diameter in each circular recesses show enhanced resistance to compression after spontaneous chondrogenesis (21-day culture) compared to those cultured for 2 days (spheroids compaction). (a) Equipment used to perform the loading tests. (b, c) One CPC spheroid between 2 plates before and after compression, respectively. Note spheroid deformation of 25% of the original diameter in (c). Five compression cycles were performed, with a load phase duration of 20 seconds and a recovery phase of 10 seconds. (d) Data collected from 3 spheroids of three different samples after spheroid compaction (2 days, light gray column) and after spontaneous chondrogenesis (21 days, dark gray column) are expressed as mean ± SD. ^∗∗∗∗p < 0.0001.

Figure 6

The CPC spheroids after spontaneous chondrogenesis are strongly positive for typical markers of cartilage tissue. (a) Hematoxylin and Eosin staining revealed rather rounded cells inside the spheroid. (b) N-cadherin is strongly present; (c–f) extracellular matrix typical markers of cartilage are shown throughout the spheroid area; (c) collagen type II; (d) collagen type VI; (e) aggrecan; (f) chondroitin sulfate. Bar size: 20 μm. (g) Control of nonspecific binding of secondary antibody (immune reaction carried out in the absence of the primary antibody). All reactions were done in the same moment. Bar size: 100 μm; μm: micrometers. (h) qPCR analysis of CPC spheroids showing upregulation of the SOX5 and SOX6 genes comparing to monolayer after 21 days of cell culture (^∗∗p < 0.01). The expression of SOX9 gene was not statistically significant (p = 0.05293). Standard errors are shown.

Figure 7

The CPC spheroids retained their fusion capacity even after spontaneous chondrogenesis. The CPC spheroids maintained for 2 days (a–c) and 21 days (d–f) in cell spheroid culture retained their fusion capacity. The CPC spheroids in close contact after 1 hour (a, d), 1 day (b, e) and 7 days (c, f). Note a complete fusion in c. Phase contrast microscopy. Bar size: 400 μm.