The expression of certain genes related to chondrogenesis, including Sox9, Col1, Col2, Acan, and Runx2, was estimated at day 10 and 21 of induction. These genes were compared to the housekeeping gene β-actin. RNA was extracted from chondrogenic spheroids and the control ones by using Easy Blue Total RNA Extraction Kit (iNtRON Biotechnology, Korea) following the manufacturer’s instruction. Quantitative RT-PCR was performed using 2x qPCRBIO SyGreen 1-Step Lo-ROX (PCRBIO Systems, London, UK) following the manufacturer’s instructions and using the gene-specific primers designed by PrimerBlast device (NCBI) (Table 1). Run parameters consisted of a single initial cycle of 45°C for 10 minutes and 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds, and 59°C for 15 seconds, and then end cycle 72°C for 15 minutes. The melting curves followed the default process of the real-time PCR device (Eppendorf, Hamburg, Germany). The expression rate of target genes of the sample was compared to the control (ADSC spheroid) by using the method of Livak: R =2^-ΔΔCt37.

Data sets were statistically analyzed by ANOVA test using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA). Differences between all the groups were considered statistically significant when the p-value was less than 0.05.

ADSCs displayed a particular morphological shape, which was an elongated shape like fibroblasts, when cultured in the plastic flasks (Figure 1A). ADSCs were verified for their differentiation potential to mesoderm-derived cells, including osteoblasts, chondrocytes and adipocytes. After induction in the osteogenic medium, ADSCs accumulated calcium in their extracellular matrix; therefore, these cells became red when stained with Alizarin red dye (Figure 1B). In the adipogenic medium, there were lipid droplets formed in cellular cytosols. These lipid droplets were stained with Oil Red O that became red (Figure 1C). Furthermore, in the chondrogenic medium, ADSCs were successfully differentiated into chondroblasts which were positive by Alcian staining assay because of the accumulation of proteoglycans (Figure 1D).

The flow cytometry results showed that the obtained ADSCs expressed high levels of CD44 (Figure 1I), CD73 (Figure 1J), CD90 (Figure 1K), and CD105 (Figure 1L), with 98.5 ± 0.46%, 98.9 ± 1.2%, 97.7 ± 2.2%, and 94.9 ± 1.8% cells staining positive, respectively. In contrast, they were virtually negative for CD14 (Figure 1F), CD34 (Figure 1G), and HLA-DR (Figure 1H), with only 0.08 ± 0.04%, 0.27 ± 0.24%, and 0.14 ± 1.0% cells staining positive, respectively.

Figure 1

ADSC spheroids were formed at a dose of 1,000 cells/drop. One day after seeding, the cells settled at the bottom of the drop by the influence of gravity and tended to clump together to form a cell mass (Figure 2), compared to the first day (Figure 2A). The masses in the hanging drops were heterogeneous in shape, and the irregular and incomplete fringe constituted a unified mass due to a few outside discrete cell clusters. However, from day 3 to day 7 (Figure 2C-D), the fringe of the cell mass was more uniform with a rounded shape.

The bar chart in Figure 2E compared the size of the spheroids between day 1 and day 7. According to the data, the diameter of spheroids significantly decreased from 252.8 ± 5.6 μm at day 1 to 234.0 ± 4.4 μm at day 2, and 210.3 ± 3.3 μm at day 3, respectively (p < 0.05). On the third day, the cell mass remained spherical and considerably expanded to 220.2 ± 3.1 μm at day 6 (p < 0.05) and 242.7 ± 2.7 μm at day 7 (p < 0.05). In summary, the ADSC-derived cell masses on the third day of formation were the smallest and densest spheroids compared with those on the other days.

Figure 2

Based on the spheroid size changes within seven days, the spheroids were stable in structure and showed signs of proliferation starting on the third day. Therefore, the assessment of cell viability and cell death was carried out on day 3 and day 7. Spheroids at each time point were distinguished between live and dead cells by the blue color of Hoechst 33258 and the red color of PI. The pictures showed that there was an accumulation of live cells only, as illustrated by the distribution of the blue signal in the spheroids on day 3 (Figure 3A and C). Additionally, there was almost no visible red signal representing dead cells in the spheroid image on day 3 (Figure 3), but a large red signal area appeared in the center of the spheroid on day 7 (Figure 3F). In the merged images, the necrotic core noticeably appeared on spheroids on day 7, in contrast to the appearance of the core on day 3.

Hematoxylin and Eosin (H&E) staining of sections of ADSC spheroids on day 3 and day 7 provided similar conclusions as the results of the viability assessment above of the ADSC spheroids (Figure 4). The morphological features of spheroid sections differed from day 3 to day 7. On the third day, cells were evenly distributed throughout the spheroid. On the seventh day, cells mostly assembled in the outer layer of the spheroid, while in the central region the cells and their nuclei were swelling with pale eosinophilic cytoplasm. These results demonstrate that necrosis occurred in the core of the spheroid.

Figure 3

Histological analysis results showed that differentiated spheroids displayed the cartilage phenotype. These spheroids were positive with Alcian blue and Safranin O/Fast green (Figure 5). The spheroids positive with Alcian blue staining assay demonstrated that stem cells inside spheroids successfully accumulated sulfated glycosaminoglycans (GAGs) of cartilage. Indeed, non-inducible spheroids were negative with this assay.

Similarly, the double-staining of Safranin O/Fast Green dyes also showed that the control sample appeared only green in color. The green of the Fast green dye is typical for non-cartilage ingredients. Meanwhile, the 21-day differentiated spheroids were positive with safranin O (Figure 5D), and hence, spheroids were dyed red. This is the sign of changes in the substrate composition of non-cartilage tissue into cartilage tissue.

Figure 5

There was a significant difference in type II collagen expression between the control (Figure 6A-D) and the induction sample (Figure 6E-H). Immunofluorescence analysis of the chondrocyte spheroids revealed the formation of type II collagen on day 21. Meanwhile, there was no sign of type II collagen expression in the control group. These result are similar to those of previously published studies (Figure 6 I-K).

The chart of the expression of genes, including Sox9, Col1, Col2, Runx2 and Acan, which are all involved in chondrogenesis and cartilage formation, is shown in Figure 7. Briefly, all genes mentioned above were upregulated during the induction spheroids compared to the control spheroids. In the early stage (day 10), the expression levels of Sox9, col1, col2, acan, and Runx2 were upregulated by 17.8 ± 6.8, 8.3 ± 3.7, 16.2 ± 5.3, 6.9 ± 1.4, and 4.8 ± 0.3-fold, respectively, though the levels were not significantly (p > 0.05) different compared to ADSC spheroids. In the later stage (day 21), the expression levels of Sox9, Col1, Col2, and Acan were remarkably increased in the chondrogenic-induced ADSC spheroids (p < 0.05), compared to the control spheoids, reaching an increase of 35.1 ± 8.6, 42.4 ± 6.0, to 25.3 ± 0.01, and 40.0 ± 1.5-fold, respectively, despite the negligible increase of Runx2 (p > 0.05).

Figure 7

Currently, there are many ways to repair cartilage damage, depending on the location and severity of the damage, age, and pathological state38. With a large area of cartilage tissue, cartilage tissue and cell transplantation are greatly effective solutions. However, these methods are limited because of dependence on tissue sources and donated cells, and donated sources are not sufficient to supply the transplanting needs. Therefore, the development of tissue engineering has brought about a promising solution combining the potential of stem cells and 3D culture to create cartilage microtissue towards regenerating tissue to respond to transplant needs to overcome the damage. In this study, we evaluated the induced, chondrogenic differentiation microtissue with respect to these characteristics: structure, matrix components, proteins, and gene expression.

In the first experiment, ADSCs were expanded and characterized. The results showed that the ADSC population possessed three characteristics of mesenchymal stem cells, following the minimal criteria and standards for defining multipotent mesenchymal stromal cells by the International Society for Cellular Therapy (ISCT), initially proposed by Dominici et al. (2006)36. Firstly, ADSC adhered on plastic when maintained in standard culture conditions. Secondly, ADSCs showed positive expression of CD44, CD73, CD90, and CD105; moreover, they were negative for CD14, CD34, and HLA-DR. Thirdly, the ADSC were capable of differentiating into mesenchymal functional cell lineages (adipocytes, osteocytes, and chondrocytes). Therefore, the ADSCs were qualified for further experiments.

Before cartilage microtissues were created, the ADSCs were cultured in non-adhesive surfaces using the hanging-drop method to form spheroids. After 24 hours, the cells came together and tended to form spheroids. However, the connection between cells was not tight such that the cell clump size mass was somewhat large. Over time, this interaction became tighter, compressing the cell mass to gradually form a uniform spherical shape, and thus leading to a decrease in size. The compression of the spheroids created an environment in which nutrient composition, oxygen concentration, and ability to transport nutrients to cells, even deep within cells, were lower than in monolayer culture. However, it did not reduce the survival of MSCs, cells that usually reside in places with lower oxygen levels than even liver or cancer cells39. Specifically, the spheroid size increased from day 3 to 7, showing signs of spheroid proliferation. However, on day 7, there were a great number of dead cells in the spheroid core; this was demonstrated by images of the spheroid sections after Hoechst and PI double-staining and H&E staining on day 7. PI is a fluorescent dye specific for double-stranded nucleic acids in apoptosis, necrosis, and fixed cells. It cannot enter normal living cell. The red color of PI dye identifie dead cells while the purple-blue color of Hoechst identifie the DNA of living and dead cells3.

Since diffusion of nutrients and metabolism decreased from the outside to the inside, the increase in size led to a nutritional deficiency within the core of the spheroid, causing cell death. Cell death occurred in increasing direction deep into the central spheroid, forming a necrotic core. Notably, this has been mentioned in a study by Murphy et al. (2017) in which it was observed that expression levels of enzymes involved in apoptosis increased as the spheroid size increased39. Furthermore, the limited diffuse range of small molecules, such as oxygen, is in the range of 150-200 μm from the outside to the inside 40. Another study showed that smaller sized spheroids had better metabolic activity and proliferation41. Therefore, the 200 μm-size spheroid at day 3 is better for the exchange and transport of oxygen, as well as nutrients, than the bigger size.

In the process of cartilage formation, the cells were induced to produce a variety of proteins that make up extracellular-specific cartilage matrix, including GAGs and aggrecan42, 43, 44. Aggrecan is an important proteoglycan of cartilage tissue by forming a gel structure that plays an intermediary role in the interaction between cartilage cells and between cartilage cells and ECM; therefore, aggrecan plays a role in tissue load capacity 45, osmotic swelling pressure42, hydraulic permeability, and deformation 46, 47. In cartilage, aggrecan is a proteoglycan that accounts for a high proportion42, 48, 49. Both aggrecan and GAG are molecules that are expressed in the early differentiation process and play a particularly important role in determining the stiffness and tolerance of cartilage tissue, typically articular cartilage tissue43, 44. In a publication from Li et al. (2018)50, the accumulation of GAG in human bone marrow derived stem cells (BMSCs) was very high, although the pellet size was larger than those seen in our study. In another study published by Futrega et al. (2015)45, GAG expression was higher in smaller- sized BMSC pellet (about 200 μm), compared to bigger ones. In the study of Futrega et al., with the same method to detect aggrecan, the accumulation showed similar results after staining of microtissue with Alcian blue45, 51, 52. Safranin O and Alcian blue are dye specific for proteoglycans 53. In our study, the presence of proteoglycan was detected by the red color after Safranin O/Fast green staining and the blue color after Alcian blue staining. The results of the double staining with Safranin O/Fast Green did not show the color of Fast Green, which is characteristic for mineralization. This indicates that on the 21^st day of differentiation, there was no hypertrophy or ossification. Therefore, the accumulation of GAG in 200 μm-sized ADSC spheroids is a good indicator of the ability of ADSCs to differentiate into cartilage tissue.

The cartilage differentiation process is regulated by transcription regulatory factors, namely Sox9, together with Sox5 and Sox6, to activate and regulate the synthesis of extracellular matrix proteins, such as collagen I, collagen II, aggrecan, and GAG43. During the differentiation induction process, the increased expression of Sox9 results in the expression of genes, including Col1, Col2, and Acan. Col1 is strongly expressed at the beginning of cartilage differentiation, in particular, in the cell aggregation stage17. The strong expression of collagen I shows the presence of chondroblasts17, 54. Type II collagen is expressed very early, right at the later stage of cell aggregation, and this expression can increase over time and throughout the differentiation process17. In fact, Type II collagen makes up the highest proportion of collagen in cartilage tissue (about 85-90%), and is synthesized by chondrocytes 55. The study of Li et al. (2018)15 on the expression of type II collagen also showed that collagen expression was high, as characterized by the collagen II antibody staining in the blue fluorescent pellets (Figure 6K). In our study, after 21 days of differentiation, the induced-MSC spheroids showed a high expression of type II collagen as well. In summary, the high expression of these molecules in ADSC spheroid after 21 days of differentiation shows that the cartilage microtissue was created with an extracellular matrix component similar to articular cartilage tissue.

In this research study, cartilage microtissue was successfully generated from spheroids composed of ADSCs. After three days of hanging drop culture, the spheroid reached a diameter of about 200 μm. After 21 days of differentiation, cartilage microtissue expressed several particular features of cartilage tissue, including accumulation of aggrecan, GAG, and type II collagen, as well as enhanced expression of Sox9, Col2, and Acan genes. These evidence support that the microtissue can be considered as a cartilage tissue that has potential applications in cartilage implants.

3D: three-dimensional

Acan: Aggrecan

ADSC: Adipose derived stem cells

CD: Cluster of differentiation

Col: Collagen

ECM: Extracellular matrix

GAGs: Glycosaminoglycans

H&E: Hematoxylin and Eosin

ISCT: International Society for Cellular Therapy

MSC: Mesenchymal stem cell

PI: Propidium iodide

Not applicable.

All authors equally contributed in this work. All authors read and approved the final manuscript.

This research is funded by the University of Science, VNU-HCM, under grant number T2018-34.

Data and materials used and/or analysed during the current study are available from the corresponding author on reasionable request.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.