Autophagy Is a Crucial Path in Chondrogenesis of Adipose-Derived Mesenchymal Stromal Cells Laden in Hydrogel

Autophagy is a cellular process that contributes to the maintenance of cell homeostasis through the activation of a specific path, by providing the necessary factors in stressful and physiological situations. Autophagy plays a specific role in chondrocyte differentiation; therefore, we aimed to analyze this process in adipose-derived mesenchymal stromal cells (ASCs) laden in three-dimensional (3D) hydrogel. We analyzed chondrogenic and autophagic markers using molecular biology, immunohistochemistry, and electron microscopy. We demonstrated that ASCs embedded in 3D hydrogel showed an increase expression of typical autophagic markers Beclin 1, LC3, and p62, associated with clear evidence of autophagic vacuoles in the cytoplasm. During ASCs chondrogenic differentiation, we showed that autophagic markers declined their expression and autophagic vesicles were rare, while typical chondrogenic markers collagen type 2, and aggrecan were significantly increased. In line with developmental animal models of cartilage, our data showed that in a 3D hydrogel, ASCs increased their autophagic features. This path is the fundamental prerequisite for the initial phase of differentiation that contributes to fueling the cells with energy and factors necessary for chondrogenic differentiation.


Introduction
Autophagy is a highly conservative eukaryotic process that represents a crucial path in the maintenance of cellular homeostasis under physiological and stressful situations. It is characterized by an intracellular degradation and recycling pathway in which metabolites generated in the vacuoles or lysosomes are reused as sources of energy or for the synthesis of new macromolecules [1]. A series of protein complexes, activated by autophagyrelated genes (atg), coordinate and regulate the formation of the autophagosome and auto-lysosome membrane [2][3][4][5][6][7]. The autophagic process starts with a UNC 51-like kinase 1 (ULK1) induction complex followed by Beclin 1 (BECN1) protein-nucleation complex that gives rise to a nascent autophagosome membrane [1,2,5]. The microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B/LC3)-complex elongate the autophagosome membrane by encapsulating the cargo in vesicles that subsequently generate an autolysosome by the action of sequestosome 1 (SQSTM1/p62)-complex [1,8]. The contents are then degraded by proteases, lipases, nucleases, and glycosidases, and the breakdown products (amino acids, lipids, nucleosides, and carbohydrates) are released into cytosol for cell-recycling pathways [1,6,7]. The main players of autophagy machinery are lysosomes and autophagosomes and, based on the cytoplasmic components inside the autophagosomes, there are three different forms of autophagy: macro-autophagy (simply called

TGFβ3 Combined with BMP6 Support the Chondrogenic Processes Well
The chondrogenic commitment of hASCs is dependent on the growth factors us the culture medium [28,29]. By using a 3D µmass model, we preliminarily checke growth factors TGFβ3 and BMP6, alone and in combination, to define the best cond for inducing hASCs chondrogenic differentiation. As shown in Figure S1 demonstrated that TGFβ3 combined with BMP6 was the most efficient conditio inducing the proteoglycan staining, as well as the COL2A1 and ACAN genes expre at day 28. According to these preliminary results, the chondrogenic differentiation of h laden in VG-RGD was induced by TGFβ3 and BMP6 and analyzed in basal condition 2) and after chondrogenic induction (days 10 and 28), as indicated in the experim scheme ( Figure 2). We confirmed, as we previously reported [34], that the COL2A1 and ACAN were not detected at day 2, while an increase was already demonstrated at day 1 significantly increased at day 28 ( Figure 3a). Interestingly, at day 28, we also confirm positive staining for collagen type 2 ( Figure 3b). The rheological and mecha properties of VG-RGD hydrogel have been previously reported [34], and these confirmed that the chondrogenic differentiation occurred following an increase i typical chondrogenic markers, as we previously reported [34].

TGFβ3 Combined with BMP6 Support the Chondrogenic Processes Well
The chondrogenic commitment of hASCs is dependent on the growth factors used in the culture medium [28,29]. By using a 3D µmass model, we preliminarily checked the growth factors TGFβ3 and BMP6, alone and in combination, to define the best conditions for inducing hASCs chondrogenic differentiation. As shown in Figure S1, we demonstrated that TGFβ3 combined with BMP6 was the most efficient condition for inducing the proteoglycan staining, as well as the COL2A1 and ACAN genes expression at day 28.
According to these preliminary results, the chondrogenic differentiation of hASCs laden in VG-RGD was induced by TGFβ3 and BMP6 and analyzed in basal condition (day 2) and after chondrogenic induction (days 10 and 28), as indicated in the experimental scheme ( Figure 2).

TGFβ3 Combined with BMP6 Support the Chondrogenic Processes Well
The chondrogenic commitment of hASCs is dependent on the growth factors used in the culture medium [28,29]. By using a 3D µmass model, we preliminarily checked the growth factors TGFβ3 and BMP6, alone and in combination, to define the best conditions for inducing hASCs chondrogenic differentiation. As shown in Figure S1, we demonstrated that TGFβ3 combined with BMP6 was the most efficient condition for inducing the proteoglycan staining, as well as the COL2A1 and ACAN genes expression at day 28.
According to these preliminary results, the chondrogenic differentiation of hASCs laden in VG-RGD was induced by TGFβ3 and BMP6 and analyzed in basal condition (day 2) and after chondrogenic induction (days 10 and 28), as indicated in the experimental scheme ( Figure 2). We confirmed, as we previously reported [34], that the COL2A1 and ACAN genes were not detected at day 2, while an increase was already demonstrated at day 10 and significantly increased at day 28 ( Figure 3a). Interestingly, at day 28, we also confirmed a positive staining for collagen type 2 ( Figure 3b). The rheological and mechanical properties of VG-RGD hydrogel have been previously reported [34], and these data confirmed that the chondrogenic differentiation occurred following an increase in the typical chondrogenic markers, as we previously reported [34]. We confirmed, as we previously reported [34], that the COL2A1 and ACAN genes were not detected at day 2, while an increase was already demonstrated at day 10 and significantly increased at day 28 ( Figure 3a). Interestingly, at day 28, we also confirmed a positive staining for collagen type 2 ( Figure 3b). The rheological and mechanical properties of VG-RGD hydrogel have been previously reported [34], and these data confirmed that the chondrogenic differentiation occurred following an increase in the typical chondrogenic markers, as we previously reported [34]. Real-time PCR analysis of COL2A1 and ACAN genes of hASCs embedded in VG-RGD hydrogel at days 2, 10, and 28 (a). Data were expressed as % GAPDH (housekeeping gene) and represented as a boxplot with median, minimum, and maximum. Kruskal-Wallis with Dunn's multiple comparisons test was used for statistical analysis: * indicates differences along time points (day 10 to day 28); *** p < 0.0005, **** p < 0.0001. Representative images of immunohistochemical analysis at day 28: negative control (control) and collagen type 2 (b). Positive areas are pink. Bar = 100 µm.

Autophagic Markers Modulation Associated with Chondrogenic Differentiation
Autophagy is a well-known catabolic process that allows the cells to destroy or recycle damaged organelles or protein to ensure their survival by protecting the cells from senescence and contributing to their rejuvenation [39,40]. It has been shown that the autophagy path is strictly correlated with chondrogenic differentiation [24,25,41]. The main autophagy players are Beclin 1, LC3, and p62, known to be essential for autophagosome formation in MSCs [42].
First, we analyzed these genes at day 2 in basal condition using molecular biology techniques. As shown in Figure 4, we demonstrated that the gene expression of SQSTM1 was significantly higher than BECN1 and MAP1LC3B, and MAP1LC3B was higher than BECN1 in hASCs embedded in VG-RGD hydrogel. A similar trend was also confirmed in hASCs maintained in 2D monolayer or in 3D µmass ( Figure S2), characterized by a significant increase of SQSTM1 in VG-RGD. The hydrogel matrix widely contributed to increasing the expression of these three autophagic genes, suggesting that the microenvironment influenced the properties of the cells, as previously reported by using other hydrogel types [43]. In fact, as we previously demonstrated [34], VG hydrogel with RGD motif created a better microenvironment than VG without RGD for ASCs chondrogenic differentiation by up-regulating the chondrogenic markers and decreasing the collagen type 1 fibrotic marker. This result represents an important prerequisite for the potential treatment of cartilage defects. It has been shown, in line with our data, that basal autophagy is high in the MSC population and is required for the maintenance of their stemness and self-renewal capabilities [44,45]. Real-time PCR analysis of COL2A1 and ACAN genes of hASCs embedded in VG-RGD hydrogel at days 2, 10, and 28 (a). Data were expressed as % GAPDH (housekeeping gene) and represented as a boxplot with median, minimum, and maximum. Kruskal-Wallis with Dunn's multiple comparisons test was used for statistical analysis: * indicates differences along time points (day 10 to day 28); *** p < 0.0005, **** p < 0.0001. Representative images of immunohistochemical analysis at day 28: negative control (control) and collagen type 2 (b). Positive areas are pink. Bar = 100 µm.

Autophagic Markers Modulation Associated with Chondrogenic Differentiation
Autophagy is a well-known catabolic process that allows the cells to destroy or recycle damaged organelles or protein to ensure their survival by protecting the cells from senescence and contributing to their rejuvenation [39,40]. It has been shown that the autophagy path is strictly correlated with chondrogenic differentiation [24,25,41]. The main autophagy players are Beclin 1, LC3, and p62, known to be essential for autophagosome formation in MSCs [42].
First, we analyzed these genes at day 2 in basal condition using molecular biology techniques. As shown in Figure 4, we demonstrated that the gene expression of SQSTM1 was significantly higher than BECN1 and MAP1LC3B, and MAP1LC3B was higher than BECN1 in hASCs embedded in VG-RGD hydrogel. A similar trend was also confirmed in hASCs maintained in 2D monolayer or in 3D µmass ( Figure S2), characterized by a significant increase of SQSTM1 in VG-RGD. The hydrogel matrix widely contributed to increasing the expression of these three autophagic genes, suggesting that the microenvironment influenced the properties of the cells, as previously reported by using other hydrogel types [43]. In fact, as we previously demonstrated [34], VG hydrogel with RGD motif created a better microenvironment than VG without RGD for ASCs chondrogenic differentiation by up-regulating the chondrogenic markers and decreasing the collagen type 1 fibrotic marker. This result represents an important prerequisite for the potential treatment of cartilage defects. It has been shown, in line with our data, that basal autophagy is high in the MSC population and is required for the maintenance of their stemness and self-renewal capabilities [44,45]. represented as a boxplot with median, minimum, and maximum. Kruskal-Wallis with Dunn's multiple comparisons test was used for statistical analysis: * indicates differences between autophagic markers; *p < 0.05, ** p < 0.005, **** p < 0.0001.
Interestingly, in line with molecular biology data, we confirmed by quantification of immunohistochemical staining a significantly high percentage of positive cells to all the autophagic markers analyzed (Beclin 1, LC3, p62) at day 2. Beclin 1 was positive in approximately 65% of the cells, while p62 and LC3 showed a significantly higher percentage (approximately 85% of positive cells). Interestingly, LC3 and p62 were both significantly higher than Beclin 1 ( Figure 5), confirming an activation of this dynamic biological process in hASCs embedded in VG-RGD hydrogel. In fact, it has been shown that modulation of autophagy (activation or deactivation) is a mechanism for controlling cell differentiation and homeostasis [1,15,23,24].  Interestingly, in line with molecular biology data, we confirmed by quantification of immunohistochemical staining a significantly high percentage of positive cells to all the autophagic markers analyzed (Beclin 1, LC3, p62) at day 2. Beclin 1 was positive in approximately 65% of the cells, while p62 and LC3 showed a significantly higher percentage (approximately 85% of positive cells). Interestingly, LC3 and p62 were both significantly higher than Beclin 1 ( Figure 5), confirming an activation of this dynamic biological process in hASCs embedded in VG-RGD hydrogel. In fact, it has been shown that modulation of autophagy (activation or deactivation) is a mechanism for controlling cell differentiation and homeostasis [1,15,23,24]. represented as a boxplot with median, minimum, and maximum. Kruskal-Wallis with Dunn's multiple comparisons test was used for statistical analysis: * indicates differences between autophagic markers; *p < 0.05, ** p < 0.005, **** p < 0.0001.
Interestingly, in line with molecular biology data, we confirmed by quantification of immunohistochemical staining a significantly high percentage of positive cells to all the autophagic markers analyzed (Beclin 1, LC3, p62) at day 2. Beclin 1 was positive in approximately 65% of the cells, while p62 and LC3 showed a significantly higher percentage (approximately 85% of positive cells). Interestingly, LC3 and p62 were both significantly higher than Beclin 1 ( Figure 5), confirming an activation of this dynamic biological process in hASCs embedded in VG-RGD hydrogel. In fact, it has been shown that modulation of autophagy (activation or deactivation) is a mechanism for controlling cell differentiation and homeostasis [1,15,23,24].  The autophagic genes were then analyzed during the hASCs chondrogenic differentiation at day 10 and day 28. At day 10, we showed a significant increase in the MAP1LC3B gene compared to BECN1, while at day 28 the MAP1LC3B and SQSTM1 genes were significantly higher than BECN1 ( Figure 6). comparisons test was used for statistical analysis: * indicates differences between autophagic markers; **** p < 0.0001.
The autophagic genes were then analyzed during the hASCs chondrogenic differentiation at day 10 and day 28. At day 10, we showed a significant increase in the MAP1LC3B gene compared to BECN1, while at day 28 the MAP1LC3B and SQSTM1 genes were significantly higher than BECN1 ( Figure 6).

Day10
Day28 0  We confirmed the same trend by immunohistochemical analysis, showing at day 10 a significant increase in not only LC3 but also in p62 proteins. Interestingly, at day 28, we found a decrease in p62 and Beclin 1 proteins compared to day 10 ( Figure 7).  We confirmed the same trend by immunohistochemical analysis, showing at day 10 a significant increase in not only LC3 but also in p62 proteins. Interestingly, at day 28, we found a decrease in p62 and Beclin 1 proteins compared to day 10 ( Figure 7). comparisons test was used for statistical analysis: * indicates differences between autophagic markers; **** p < 0.0001.
The autophagic genes were then analyzed during the hASCs chondrogenic differentiation at day 10 and day 28. At day 10, we showed a significant increase in the MAP1LC3B gene compared to BECN1, while at day 28 the MAP1LC3B and SQSTM1 genes were significantly higher than BECN1 ( Figure 6).

Day10
Day28 0  We confirmed the same trend by immunohistochemical analysis, showing at day 10 a significant increase in not only LC3 but also in p62 proteins. Interestingly, at day 28, we found a decrease in p62 and Beclin 1 proteins compared to day 10 ( Figure 7).  Moreover, considering the high percentage of positive cells found at day 2, we showed a decrease at days 10 and 28 in Beclin 1 that, starting from approximately 65% of positive cells, reached a level lower than 20% of positive cells (Figure 8). Interestingly, also p62 and LC3 decreased, but in a lower percentage, from day 2 to day 10 ( Figure 8). Finally, we focused on the intensity of the positive cells, and we observed that Beclin 1 immunostaining intensity was higher at day 2 than day 28. Interestingly, although the percentages of LC3and p62-positive cells were comparable at day 2 and day 28 (Figure 8), we noticed a lower intensity of LC3 than p62 at day 2 and day 28 (Figures 5 and 7). It is known that autophagy is a dynamic modulated process that is enhanced by cells in specific conditions for increasing their energy and promoting the biomacromolecule synthesis necessary for maintaining cell survival and homeostasis [1,5,9,23]. When chondrogenic differentiation occurs (at days 10 and 28), we showed a decrease in Beclin 1, a known starting sensor of the autophagic process associated with a down-modulation of LC3 and p62 responsible for autophagosome maturation, confirming the regulatory role of this process.
Moreover, considering the high percentage of positive cells found at day 2, we showed a decrease at days 10 and 28 in Beclin 1 that, starting from approximately 65% of positive cells, reached a level lower than 20% of positive cells (Figure 8). Interestingly, also p62 and LC3 decreased, but in a lower percentage, from day 2 to day 10 ( Figure 8). Finally, we focused on the intensity of the positive cells, and we observed that Beclin 1 immunostaining intensity was higher at day 2 than day 28. Interestingly, although the percentages of LC3-and p62-positive cells were comparable at day 2 and day 28 ( Figure  8), we noticed a lower intensity of LC3 than p62 at day 2 and day 28 (Figures 5 and 7). It is known that autophagy is a dynamic modulated process that is enhanced by cells in specific conditions for increasing their energy and promoting the biomacromolecule synthesis necessary for maintaining cell survival and homeostasis [1,5,9,23]. When chondrogenic differentiation occurs (at days 10 and 28), we showed a decrease in Beclin 1, a known starting sensor of the autophagic process associated with a down-modulation of LC3 and p62 responsible for autophagosome maturation, confirming the regulatory role of this process. Transmission electron microscopy analysis helped to confirm and better describe the nature of autophagic vacuoles that were also previously evaluated by immunohistochemical analysis. At day 2 (Figure 9), ultrastructural analysis of hASCs embedded in VG-RGD showed an oval, round, or elongated cell morphology. Nuclei were large, slightly indented, with dispersed chromatin (c). Cytoplasm revealed abundant rough endoplasmic reticulum (rer), some lipid droplets (ld), numerous well-preserved mitochondria (m), with regularly orientated cristae. We observed a high concentration of vacuoles (v) that differed for morphology and heterogenous intraluminal contents. These characteristics are ascribable to phagocytized cytoplasmic material. We also noted the presence of some empty vacuoles (ev). Different autophagic vacuoles contained partially degraded material compatible with late autophagic vacuoles (lv), while the doublemembraned (black arrow) vacuoles containing cytoplasmic material slated to be degraded, ascribable to autophagosome (AP), were observed less. All together, these results demonstrate that all the autophagic markers analyzed (Beclin 1, LC3, p62,) are highly expressed and associated with an increase in autophagic vacuoles into the cells before starting the chondrogenic differentiation, suggesting that the cells activate this process for the maintenance of cell homeostasis in the 3D hydrogel. It has been shown [43] that rabbit chondrocytes seeded on different hydrogels show an increase in autophagyrelated genes on gellan gum and agarose hydrogels, indicating that these polysaccharides activate this process, as we found embedding hASCs in VG-RGD hydrogel.
After 10 days of chondrogenic differentiation (Figure 9), we noted in the ASCs the presence of elongated nuclei with highly dispersed chromatin (c) and prominent Transmission electron microscopy analysis helped to confirm and better describe the nature of autophagic vacuoles that were also previously evaluated by immunohistochemical analysis. At day 2 (Figure 9), ultrastructural analysis of hASCs embedded in VG-RGD showed an oval, round, or elongated cell morphology. Nuclei were large, slightly indented, with dispersed chromatin (c). Cytoplasm revealed abundant rough endoplasmic reticulum (rer), some lipid droplets (ld), numerous well-preserved mitochondria (m), with regularly orientated cristae. We observed a high concentration of vacuoles (v) that differed for morphology and heterogenous intraluminal contents. These characteristics are ascribable to phagocytized cytoplasmic material. We also noted the presence of some empty vacuoles (ev). Different autophagic vacuoles contained partially degraded material compatible with late autophagic vacuoles (lv), while the double-membraned (black arrow) vacuoles containing cytoplasmic material slated to be degraded, ascribable to autophagosome (AP), were observed less. All together, these results demonstrate that all the autophagic markers analyzed (Beclin 1, LC3, p62,) are highly expressed and associated with an increase in autophagic vacuoles into the cells before starting the chondrogenic differentiation, suggesting that the cells activate this process for the maintenance of cell homeostasis in the 3D hydrogel. It has been shown [43] that rabbit chondrocytes seeded on different hydrogels show an increase in autophagy-related genes on gellan gum and agarose hydrogels, indicating that these polysaccharides activate this process, as we found embedding hASCs in VG-RGD hydrogel.
After 10 days of chondrogenic differentiation (Figure 9), we noted in the ASCs the presence of elongated nuclei with highly dispersed chromatin (c) and prominent nucleolus (data not shown). Cytoplasm was characterized by numerous well-preserved mitochondria, many lipid droplets (ld), and an extended rough endoplasmic reticulum (rer) composed of long, irregular, and thin cisternae that demonstrated an active protein synthesis. We observed a decrease in the number of autophagic vacuoles inside the cytoplasm, which could be ascribable to late autophagic vacuoles (lv). In this chondrogenic differentiation phase, we showed banded collagen fibrils (coll) distributed randomly and not oriented throughout the extracellular space.
After 28 days of differentiation (Figure 9), the chondrogenic cells were arranged in a small group, and we showed the presence of abundant deposition of banded collagen fibrils (coll) around them, suggesting that the differentiation process was happening. At this time point, the presence of autophagic vesicles was strongly lower compared to differentiated cells at day 10. The cells observed were characterized by slightly indented nuclei with dispersed chromatin (c), prominent nucleolus (nu), abundant and dilated rough endoplasmic reticulum (rer), lipid droplets (ld), many mitochondria, and numerous transport vesicles (tv). These features confirmed that the hASCs encapsulated in VG-RGD were strongly committed to protein synthesis and secretion.
nucleolus (data not shown). Cytoplasm was characterized by numerous well-preserved mitochondria, many lipid droplets (ld), and an extended rough endoplasmic reticulum (rer) composed of long, irregular, and thin cisternae that demonstrated an active protein synthesis. We observed a decrease in the number of autophagic vacuoles inside the cytoplasm, which could be ascribable to late autophagic vacuoles (lv). In this chondrogenic differentiation phase, we showed banded collagen fibrils (coll) distributed randomly and not oriented throughout the extracellular space.
After 28 days of differentiation (Figure 9), the chondrogenic cells were arranged in a small group, and we showed the presence of abundant deposition of banded collagen fibrils (coll) around them, suggesting that the differentiation process was happening. At this time point, the presence of autophagic vesicles was strongly lower compared to differentiated cells at day 10. The cells observed were characterized by slightly indented nuclei with dispersed chromatin (c), prominent nucleolus (nu), abundant and dilated rough endoplasmic reticulum (rer), lipid droplets (ld), many mitochondria, and numerous transport vesicles (tv). These features confirmed that the hASCs encapsulated in VG-RGD were strongly committed to protein synthesis and secretion. The immunohistochemical analysis and electron microscopy evaluations helped demonstrate that when the typical chondrogenic markers, such as collagen type 2 and aggrecan were increased (day 28, Figure 3), a significant decrease in Beclin 1, LC3, and p62 autophagic markers was observed, indicating that autophagy modulation in hASCs affects their ability to differentiate and suggesting an interplay between the two processes ( Figure 10). It has been shown, in osteogenic differentiation [46], that transient autophagy The immunohistochemical analysis and electron microscopy evaluations helped demonstrate that when the typical chondrogenic markers, such as collagen type 2 and aggrecan were increased (day 28, Figure 3), a significant decrease in Beclin 1, LC3, and p62 autophagic markers was observed, indicating that autophagy modulation in hASCs affects their ability to differentiate and suggesting an interplay between the two processes ( Figure 10). It has been shown, in osteogenic differentiation [46], that transient autophagy activation supplies cells with energy, confirming that autophagy is fundamental in the initial phase because it fuels the cells that are then prone to differentiation. Interestingly, this evidence is also in line with papers [12,13] that studied the developmental of cartilage using in vivo animal models, demonstrating that the initial induction of autophagy played a peculiar role in coordinating chondrocyte differentiation and pre-chondrogenic cells transitioning into chondroblasts. Moreover, it has been shown that autophagy protects the cells from senescence [39], contributes to their rejuvenation [40], and shows a potential key contribution to hASCs' therapeutic action [20,47] by providing a vital activity to cells. In fact, it has also been postulated that there is a relationship between autophagy and some signals that regulate the transduction mechanism directly involved in their differentiation [48], such as wingless/integrated (Wnt)/β-catenin, neurogenic locus notch homolog protein (Notch), and NF-E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) signaling. All these autophagy functions on cells might represent an interesting way to improve the differentiation of hASCs for purposes of cartilage regeneration.
activation supplies cells with energy, confirming that autophagy is fundamental in the initial phase because it fuels the cells that are then prone to differentiation. Interestingly, this evidence is also in line with papers [12,13] that studied the developmental of cartilage using in vivo animal models, demonstrating that the initial induction of autophagy played a peculiar role in coordinating chondrocyte differentiation and pre-chondrogenic cells transitioning into chondroblasts. Moreover, it has been shown that autophagy protects the cells from senescence [39], contributes to their rejuvenation [40], and shows a potential key contribution to hASCs' therapeutic action [20,47] by providing a vital activity to cells. In fact, it has also been postulated that there is a relationship between autophagy and some signals that regulate the transduction mechanism directly involved in their differentiation [48], such as wingless/integrated (Wnt)/β-catenin, neurogenic locus notch homolog protein (Notch), and NF-E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) signaling. All these autophagy functions on cells might represent an interesting way to improve the differentiation of hASCs for purposes of cartilage regeneration.

Conclusions
In conclusion, we demonstrated that chondrogenic differentiation of hASCs embedded in 3D hydrogel is associated to a significant inhibition of autophagy vesicles and markers. The hydrogel environment contributed to increasing the hASCs' autophagy markers before starting chondrogenesis, thus furnishing the building blocks necessary for the chondrogenic differentiation of hASCs by acting on specific cellular processes. However, a limitation of this study is the importance of a deep analysis of VG-RGD hydrogel as a modulator of ASC characteristics that could help to identify a new positive pathway of chondrogenesis. Autophagy modulation might represent a new perspective for enhancing or repressing the characteristic of hASCs, and thus opening a new pathway in cartilage regeneration. Hydrogel's properties are an intriguing way to guide the differentiation of ASCs by facilitating their translation to the clinic.

Conclusions
In conclusion, we demonstrated that chondrogenic differentiation of hASCs embedded in 3D hydrogel is associated to a significant inhibition of autophagy vesicles and markers. The hydrogel environment contributed to increasing the hASCs' autophagy markers before starting chondrogenesis, thus furnishing the building blocks necessary for the chondrogenic differentiation of hASCs by acting on specific cellular processes. However, a limitation of this study is the importance of a deep analysis of VG-RGD hydrogel as a modulator of ASC characteristics that could help to identify a new positive pathway of chondrogenesis. Autophagy modulation might represent a new perspective for enhancing or repressing the characteristic of hASCs, and thus opening a new pathway in cartilage regeneration. Hydrogel's properties are an intriguing way to guide the differentiation of ASCs by facilitating their translation to the clinic.

hASCs Embedding in Hydrogel
We mixed 2 × 10 6 hASCs in 1 mL of VitroGel ® -RGD hydrogel (VG-RGD, The Well Biosciences, North Brunswick, NJ, USA), diluted 1:2, prepared according to the protocol guidelines recommended by the company and previously described [34]. Briefly, 300 µL of the hydrogel suspension were gently plated into the cell crown (Scaffdex, Finland), which were transferred into 24-well plates for ionic crosslinking. Hydrogels were maintained in basal chondrogenic medium. At days 2 and 7 the cells were retrieved from the hydrogels with VitroGel ® cell recovery solution (The Well Bioscience) and analyzed by FACS to check the markers CD73, CD90, CD105, and CD 146 (BD, Franklin Lakes, NJ, USA), as previously reported [34].
HASCs embedded in VG-RGD were treated with a chondrogenic medium containing the chondrogenic factors TGF-β3 (10 ng/mL) and BMP6 (10 ng/mL). The cell culture medium was changed three times a week. Each construct was analyzed on days 2, 10, and 28 to test the chondrogenic and autophagic markers, as indicated in the experimental scheme ( Figure 2).

Molecular Biology
Total RNA was extracted from all chondrogenic samples at days 2, 10, and 28. Hydrogelladen hASCs, µmasses, and hASCs in monoculture were treated with 1 mL of Eurogold RnaPure (EuroClone), immediately flash frozen in liquid nitrogen (−196 • C) and stored in a freezer at −80 • C. RNA extraction was performed by homogenizing samples and following the Eurogold manufacturer's instructions, as previously described [49]. Reverse transcription was performed using a Super Script ® Vilo™ cDNA synthesis Kit (Life Technologies), according to the manufacturer's instructions. Real-time polymerase chain reaction (PCR) was performed with QuantStudio1 (Applied Biosystems by Thermo Fisher Scientific, 0706 Singapore 739256) for the quantification of the following genes: Beclin 1 (BECN1), microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), sequestosome 1 (SQSTM1), collagen type 2 alpha 1 chain (COL2A1), aggrecan (ACAN) ( Table S1). All primer efficiency was confirmed to be high (>90%) and comparable (Table S1). For each target gene, mRNA levels were calculated, normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) according to the formula 2 −∆Ct , and expressed as a percentage of the reference gene.
The analysis was performed using red/green/blue (RGB) with Software NIS-Elements and Eclipse 90i microscope (Nikon Instruments Europe BV). Briefly, we acquired the total number of blue stained nuclei and the total number of positive stained red cells. The data were expressed as a percentage of cells positive for Beclin 1, LC3, and p62.

Transmission Electron Microscopy
For ultrastructural evaluation, the VG-RGD hydrogel-embedded hASCs were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 1 h at room temperature and for 3 h at 4 • C. Afterwards, samples were postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer for 2 h at 4 • C, dehydrated in an ethanol series, infiltrated with propylene oxide, and embedded in Epon resin. Cross-sections of each hydrogel were cut to allow internal analysis. Ultrathin sections (80 nm thick) were stained with uranyl acetate and lead citrate (15 min each) and observed with a Jeol Jem 1011 transmission electron microscope (Jeol Jem, USA), operated at 100 kV. Images were captured using an Olympus digital camera and iTEM software.

Statistical Analysis
Statistical analysis was completed using CSS Statistical Software (Statsoft Inc., Tulsa, OK, USA). Non-parametric tests were performed since the data did not have normal or strongly asymmetric distribution. The Kruskal-Wallis with Dunn's multiple comparisons test was used, and values of p < 0.05 were considered statistically significant. Values were expressed either as the median and minimum and maximum or as mean ± SD.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/gels8120766/s1, Figure S1: Safranin immunohistochemical staining and real-time PCR analysis of COL2A1 and ACAN genes expression of hASCs in 3D µmasses; Figure  S2: Real-time PCR analysis of BECN1, MAP1LC3B, SQSTM1 genes on hASCs in 2D monolayer, 3D µmasses and VG-RGD; Table S1: Oligonucleotide primers used for real-time PCR. Institutional Review Board Statement: Ethical review and approval were waived for this study because the human ASCs were purchased from Lonza.

Informed Consent Statement:
Not applicable because the human ASCs were purchased from Lonza.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare that there is no conflict of interest.