Oxytocin Modulates Osteogenic Commitment in Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (hASCs) are commonly harvested in minimally invasive contexts with few ethical concerns, and exhibit self-renewal, multi-lineage differentiation, and trophic signaling that make them attractive candidates for cell therapy approaches. The identification of natural molecules that can modulate their biological properties is a challenge for many researchers. Oxytocin (OXT) is a neurohypophyseal hormone that plays a pivotal role in the regulation of mammalian behavior, and is involved in health and well-being processes. Here, we investigated the role of OXT on hASC proliferation, migratory ability, senescence, and autophagy after a treatment of 72 h; OXT did not affect hASC proliferation and migratory ability. Moreover, we observed an increase in SA-β-galactosidase activity, probably related to the promotion of the autophagic process. In addition, the effects of OXT were evaluated on the hASC differentiation ability; OXT promoted osteogenic differentiation in a dose-dependent manner, as demonstrated by Alizarin red staining and gene/protein expression analysis, while it did not affect or reduce adipogenic differentiation. We also observed an increase in the expression of autophagy marker genes at the beginning of the osteogenic process in OXT-treated hASCs, leading us to hypothesize that OXT could promote osteogenesis in hASCs by modulating the autophagic process.


Introduction
Oxytocin (OXT) was the first peptide molecule to be biochemically identified [1]. OXT is a nonapeptide containing one internal disulfide bond; OXT, synthesized by the supraoptic nucleus and by the paraventricular nucleus of the hypothalamus, is carried along the axon to the posterior pituitary and released into the blood circulation [2]. OXT can be synthesized and released by many peripheral organs, such as the heart [3], uterus [4], and thymus [5]. OXT acts through its receptor (OXTR), which belongs to the heptahelical G protein-coupled receptor family, and it is expressed in many different types of tissues, such as nervous, reproductive, and immune systems [2], as well as in many cell types as osteoblasts and adipocytes [6][7][8][9].
OXT has many properties and functions related to a wide range of physiological fields, like lactation and parturition [10], energy metabolism [11], immune system [12,13], or thermoregulation and body composition [14,15]. All of these, and many other functions, influence health and well-being. . hASCs were seeded at 3500 cells/cm 2 and treated with OXT at 100, 500, and 1000 nM; untreated cells were used as control (CTR). Cells were detected under bright field illumination using a Leica Labovert FS Inverted Microscope and Leica MC170 HD Imaging System Camera. Scale bars: 100 µm. (b) OXTR mRNA expression in CTR and OXT-treated hASCs. The mRNA expression of OXTR was assessed in hASCs after 72 h of basal culture condition (CTR) or after 72 h of OXT 1000 nM treatment. Data obtained by quantitative Real-Time PCR (qPCR) were normalized using three reference genes (TATA Binding Protein-TBP, Glyceraldehyde-3-Phosphate Dehydrogenase-GAPDH and Hypoxanthine Phosphoribosyltransferase 1-HPRT1); the normalized expression value of CTR cells was set to 1, and OXTR gene expression value of OXTtreated cells was reported to CTR value. The graphic represents the normalized fold change ± standard error of the mean (SEM); n = 3; * p < 0.05.

Effects of Oxytocin on hASC Viability
To evaluate the effects of different OXT concentrations (100, 500, and 1000 nM) on hASC viability, cells were counted 72 h after OXT treatment in the presence of the Erythrosine B dye that allows discriminating between dead (red) and living cells . hASCs were seeded at 3500 cells/cm 2 and treated with OXT at 100, 500, and 1000 nM; untreated cells were used as control (CTR). Cells were detected under bright field illumination using a Leica Labovert FS Inverted Microscope and Leica MC170 HD Imaging System Camera. Scale bars: 100 µm. (b) OXTR mRNA expression in CTR and OXT-treated hASCs. The mRNA expression of OXTR was assessed in hASCs after 72 h of basal culture condition (CTR) or after 72 h of OXT 1000 nM treatment. Data obtained by quantitative Real-Time PCR (qPCR) were normalized using three reference genes (TATA Binding Protein-TBP, Glyceraldehyde-3-Phosphate Dehydrogenase-GAPDH and Hypoxanthine Phosphoribosyltransferase 1-HPRT1); the normalized expression value of CTR cells was set to 1, and OXTR gene expression value of OXT-treated cells was reported to CTR value. The graphic represents the normalized fold change ± standard error of the mean (SEM); n = 3; * p < 0.05.

Effects of Oxytocin on hASC Viability
To evaluate the effects of different OXT concentrations (100, 500, and 1000 nM) on hASC viability, cells were counted 72 h after OXT treatment in the presence of the Erythrosine B dye that allows discriminating between dead (red) and living cells (unstained). The percentage of living cells for both CTR and OXT-treated cells was comparable, indicating that OXT was not toxic for hASCs at the tested concentrations (Table 1). OXT 100 nM, OXT 500 nM, and OXT 1000 nM: hASCs treated with OXT at concentrations of 100, 500, and 1000 nM, respectively. Data are expressed as the mean percentage of living cells ± standard deviation (SD); n = 3.

Effects of Oxytocin on hASC Proliferation
Since the cell count showed comparable viability between hASCs treated with OXT and CTR cells, to evaluate the effects of OXT on hASC proliferation, a Bromodeoxyuridine (BrdU) assay was performed. hASC samples (CTR and treated cells with OXT at 100, 500, and 1000 nM for 72 h) were incubated with a BrdU labeling solution. The resulting amount of positive BrdU cells in CTR was comparable with the amount in OXT-treated cells, and no significant differences were revealed between samples (Figure 2a). To confirm proliferation results, the expression of genes involved in cell proliferation and cell cycle control was investigated in both CTR and treated hASCs only with 1000 nM OXT for 72 h. The investigated genes were markers of proliferation Ki-67 (MKI67), cyclin D1 (CCND1), cyclin-dependent kinase inhibitor 2A (CDKN2A, alias p16 INK4a ), cyclin-dependent kinase inhibitor 1A (CDKN1A, alias p21) and tumor protein p53 (TP53). Data obtained did not show any significant differences in the expression of all investigated genes between CTR and OXTtreated hASCs, thus confirming that OXT did not affect hASC proliferation when used at this concentration ( Figure 2b).
(unstained). The percentage of living cells for both CTR and OXT-treated cells was comparable, indicating that OXT was not toxic for hASCs at the tested concentrations (Table 1).

Effects of Oxytocin on hASC Proliferation
Since the cell count showed comparable viability between hASCs treated with OXT and CTR cells, to evaluate the effects of OXT on hASC proliferation, a Bromodeoxyuridine (BrdU) assay was performed. hASC samples (CTR and treated cells with OXT at 100, 500, and 1000 nM for 72 h) were incubated with a BrdU labeling solution. The resulting amount of positive BrdU cells in CTR was comparable with the amount in OXT-treated cells, and no significant differences were revealed between samples (Figure 2a). To confirm proliferation results, the expression of genes involved in cell proliferation and cell cycle control was investigated in both CTR and treated hASCs only with 1000 nM OXT for 72 h. The investigated genes were markers of proliferation Ki-67 (MKI67), cyclin D1 (CCND1), cyclin-dependent kinase inhibitor 2A (CDKN2A, alias p16 INK4a ), cyclin-dependent kinase inhibitor 1A (CDKN1A, alias p21) and tumor protein p53 (TP53). Data obtained did not show any significant differences in the expression of all investigated genes between CTR and OXTtreated hASCs, thus confirming that OXT did not affect hASC proliferation when used at this concentration ( Figure 2b).  (a) Bromodeoxyuridine (BrdU) assay was used to assess cell proliferation in untreated (CTR) or treated cells with OXT (100, 500, and 1000 nM) for 72 h. Histograms represent the mean percentage of BrdU incorporation normalized to CTR ± standard deviation (SD); n = 3. (b) Expression of cell cycle and proliferation marker genes in CTR and in 1000 nM OXT-treated hASCs. The expression of marker of proliferation Ki-67 (MKI67), cyclin D1 (CCND1), cyclin-dependent kinase inhibitor 2A (CDKN2A, alias p16 INK4a ), cyclin-dependent kinase inhibitor 1A (CDKN1A, alias p21) and tumor protein p53 (TP53) genes was assessed by quantitative Real-Time PCR (qPCR) after 72 h of OXT treatment. Data were normalized using four reference genes (Ribosomal Protein L13a-RPL13a, TATA Binding Protein-TBP, Glyceraldehyde-3-Phosphate Dehydrogenase-GAPDH, Hypoxanthine Phosphoribosyltransferase 1-HPRT1); for each gene, the normalized expression value of CTR was set up to 1, and the gene expression value of the OXT treated sample was reported to CTR value. Data are reported as normalized fold change ± standard error of the mean (SEM); n = 3.

Effects of Oxytocin on hASC Migration Ability
To assess the effect of OXT on another biological property of hASCs, we investigated the role of OXT on their migratory capacity. Scratch wound healing assay was performed in hASCs after treating them with or without OXT at 100, 500, and 1000 nM for 24 h. As shown in Figure 3, cells treated with OXT showed no difference in the ability to close the scratch "wound" compared to CTR cells for all the investigated conditions ( Figure 3); these findings were consistent with the proliferation data reported above.

Effects of Oxytocin on hASC Migration Ability
To assess the effect of OXT on another biological property of hASCs, we investigated the role of OXT on their migratory capacity. Scratch wound healing assay was performed in hASCs after treating them with or without OXT at 100, 500, and 1000 nM for 24 h. As shown in Figure 3, cells treated with OXT showed no difference in the ability to close the scratch "wound" compared to CTR cells for all the investigated conditions ( Figure 3); these findings were consistent with the proliferation data reported above. . Scratch wound healing assay in human adipose-derived stem cells (hASCs) treated or untreated with Oxytocin (OXT). The migratory ability of hASCs to close the wound in untreated (CTR) and in OXT (100, 500, and 1000 nM) treated cells were analyzed after a treatment of 24 h. (a) Images were acquired immediately after scratch (0 h) and at the indicated time-points (12, 24, 36 h) using the Leica Labovert FS Inverted Microscope equipped with a Leica MC170 HD Imaging System Camera. Scale bars: 100 µm. (b) Evaluation of the percentage of wound closure at different time points (0, 12, 24, 36 h) in CTR and OXT-treated cells. The scratch area was measured using the ImageJ software 1.53e. The results are presented as the mean percentage of wound closure normalized on the CTR cells ± standard error of the mean (SEM). The percentage of wound closure at t = 0 h was set to 0 for both CTR and OXT-treated cells; n = 3.

Effects of Oxytocin on hASC Senescence-Associated-β-Galactosidase (SA-β-Gal) Activity and Autophagy
To evaluate whether OXT could modulate hASC senescence, the SA-β-Gal staining assay was performed in untreated (CTR) or in treated hASCs with OXT (100, 500, and 1000 nM) for 72 h. The number of positively blue-stained cells, calculated as the percentage of total number of cells, was significantly higher in the treated cells compared to CTR at each OXT concentration investigated (Figure 4a,b). Therefore, we investigated the expression The scratch area was measured using the ImageJ software 1.53e. The results are presented as the mean percentage of wound closure normalized on the CTR cells ± standard error of the mean (SEM). The percentage of wound closure at t = 0 h was set to 0 for both CTR and OXT-treated cells; n = 3.

Effects of Oxytocin on hASC Senescence-Associated-β-Galactosidase (SA-β-Gal) Activity and Autophagy
To evaluate whether OXT could modulate hASC senescence, the SA-β-Gal staining assay was performed in untreated (CTR) or in treated hASCs with OXT (100, 500, and 1000 nM) for 72 h. The number of positively blue-stained cells, calculated as the percentage of total number of cells, was significantly higher in the treated cells compared to CTR at each OXT concentration investigated (Figure 4a,b). Therefore, we investigated the expression of marker genes involved in cell senescence telomerase reverse transcriptase (TERT) and BMI1 proto-oncogene, polycomb ring finger (BMI-1) in hASCs treated for 72 h with OXT at 1000 nM, the dose in which the most evident effect on SA-β-Gal activity was observed. Data reported in Figure 4c showed that OXT did not affect the expression of both TERT and BMI-1. The increase in SA-β-Gal activity is not only associated with cellular senescence, but may be related to other cellular pathways in which the lysosomal activity is involved, such as autophagy [37,38]. Thus, the expression levels of two autophagic marker genes, Beclin 1 (BECN1) and microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A), were investigated by qPCR in untreated or 1000 nM OXT-treated hASCs for 72 h. Data shown in Figure 4d indicated that 1000 nM OXT induced an increase in the gene expression of BECN1 and, significantly, of MAP1LC3A in hASCs, compared to CTR, indicating a possible involvement of OXT in the autophagy pathway ( Figure 4d). BMI1 proto-oncogene, polycomb ring finger (BMI-1) in hASCs treated for 72 h with OXT at 1000 nM, the dose in which the most evident effect on SA-β-Gal activity was observed. Data reported in Figure 4c showed that OXT did not affect the expression of both TERT and BMI-1. The increase in SA-β-Gal activity is not only associated with cellular senescence, but may be related to other cellular pathways in which the lysosomal activity is involved, such as autophagy [37,38]. Thus, the expression levels of two autophagic marker genes, Beclin 1 (BECN1) and microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A), were investigated by qPCR in untreated or 1000 nM OXT-treated hASCs for 72 h. Data shown in Figure 4d indicated that 1000 nM OXT induced an increase in the gene expression of BECN1 and, significantly, of MAP1LC3A in hASCs, compared to CTR, indicating a possible involvement of OXT in the autophagy pathway ( Figure 4d).  proto-oncogene, polycomb ring finger (BMI-1) and telomerase reverse transcriptase (TERT) genes in hASCs CTR or treated with 1000 nM OXT for 72 h. Data obtained by quantitative Real-Time PCR (qPCR) were normalized using three reference genes (TATA Binding Protein-TBP, Glyceraldehyde-3-Phosphate Dehydrogenase-GAPDH, and Hypoxanthine Phosphoribosyltransferase 1-HPRT1). (d) Expression of Beclin 1 (BECN1) and microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A) genes in hASCs CTR or treated with 1000 nM OXT for 72 h. Data obtained by qPCR were normalized using three reference genes (TBP, GAPDH, and HPRT1). In (c,d), in each graph and for each gene, the normalized expression values of CTR cells were set up to 1, and all other gene expression values were reported to that sample. Data are reported as normalized fold change ± standard error of the mean (SEM); n = 3; * p < 0.05.

Effects of Oxytocin on the Adipogenic and Osteogenic Potential of hASCs
The effects of OXT on hASC differentiation ability were also evaluated; in particular, we focused on adipogenic and osteogenic commitment. The expression of peroxisome proliferator-activated receptor gamma (PPARγ) and RUNX family transcription factor 2 (RUNX2) genes, two important adipogenic and osteogenic markers, respectively, was analyzed in hASCs after only 72 h of OXT treatment to evaluate whether OXT alone could address hASCs towards a specific commitment, in the absence of specific induction media. The expression of both PPARγ and RUNX2 genes was not altered in hASCs treated with OXT 1000 nM compared to CTR cells, suggesting that this peptide alone was not able to induce adipogenic and osteogenic differentiation ( Figure 5).
were reported to that sample. Data are reported as normalized fold change ± standard error of the mean (SEM); n = 3; * p < 0.05.

Effects of Oxytocin on the Adipogenic and Osteogenic Potential of hASCs
The effects of OXT on hASC differentiation ability were also evaluated; in particular, we focused on adipogenic and osteogenic commitment. The expression of peroxisome proliferator-activated receptor gamma (PPARγ) and RUNX family transcription factor 2 (RUNX2) genes, two important adipogenic and osteogenic markers, respectively, was analyzed in hASCs after only 72 h of OXT treatment to evaluate whether OXT alone could address hASCs towards a specific commitment, in the absence of specific induction media. The expression of both PPARγ and RUNX2 genes was not altered in hASCs treated with OXT 1000 nM compared to CTR cells, suggesting that this peptide alone was not able to induce adipogenic and osteogenic differentiation ( Figure 5). hASCs were treated for the whole duration of the adipogenic protocol with OXT 30, 100, 500, and 1000 nM; after 14 days, a significant reduction in adipogenic commitment was observed in hASCs cultured in adipogenic differentiation medium and treated with 1000 nM OXT, compared to CTR cells (Figure 6a,b). Moreover, PPARγ gene expression was investigated in CTR and in 1000 nM OXT-treated hASCs at 3, 7, and 14 days from the beginning of the adipogenic differentiation protocol. Consistent with O.R.O staining Figure 5. Expression of peroxisome proliferator-activated receptor gamma (PPARγ) and RUNX family transcription factor 2 (RUNX2) genes in human adipose-derived stem cells (hASCs) treated or untreated with Oxytocin (OXT) for 72 h. hASCs were cultured without (CTR) or with 1000 nM OXT for 72 h, and the expression of PPARγ and RUNX2 genes were assessed. Data obtained by quantitative Real-Time PCR (qPCR) were normalized using three reference genes (TATA Binding Protein-TBP, Glyceraldehyde-3-Phosphate Dehydrogenase-GAPDH, and Hypoxanthine Phosphoribosyltransferase 1-HPRT1). For each gene, the normalized expression value of CTR was set up to 1, and other gene expression values were reported to that sample. Data were reported as normalized fold change ± standard error of the mean (SEM); n = 3.

Effects of Oxytocin on the hASC Adipogenic Commitment
The effects of OXT on hASC adipogenic commitment were analyzed by culturing cells with OXT in the adipogenic induction medium for the entire period of the differentiation protocol. Adipogenesis was monitored by counting the number of the Oil Red O (O.R.O) positive cells, as well as by evaluating the expression of the adipogenic gene marker PPARγ.
hASCs were treated for the whole duration of the adipogenic protocol with OXT 30, 100, 500, and 1000 nM; after 14 days, a significant reduction in adipogenic commitment was observed in hASCs cultured in adipogenic differentiation medium and treated with 1000 nM OXT, compared to CTR cells (Figure 6a,b). Moreover, PPARγ gene expression was investigated in CTR and in 1000 nM OXT-treated hASCs at 3, 7, and 14 days from the beginning of the adipogenic differentiation protocol. Consistent with O.R.O staining results, PPARγ gene expression was statistically reduced in OXT-treated cells compared to CTR cells after 3 days from the beginning of adipogenesis induction, consistent with the knowledge that PPARγ is an early adipogenic marker gene (Figure 6c). results, PPARγ gene expression was statistically reduced in OXT-treated cells compared to CTR cells after 3 days from the beginning of adipogenesis induction, consistent with the knowledge that PPARγ is an early adipogenic marker gene (Figure 6c).

Effects of Oxytocin on the hASC Osteogenic Commitment
The effects of OXT on hASC osteogenic commitment were analyzed by culturing cells with OXT in the induction medium for the whole duration of the differentiation protocol. Osteogenesis was monitored by evaluating the amount of the Alizarin Red S dye, the mRNA expression of the osteogenic gene marker bone gamma-carboxyglutamate protein (BGLAP, also named osteocalcin) as well as the protein expression of the RUNX2, osteopontin (OPN), type 1 collagen and fibronectin.
In the first experiment, four OXT concentrations (30, 100, 500, and 1000 nM) were used during the entire osteogenic protocol. The Alizarin Red S staining, which specifically binds calcium deposits, revealed a dose-dependent increase in the osteogenic commitment in cells cultured in an osteogenic medium (OM) and treated with OXT, especially at the concentration of 1000 nM (Figure 7a). Thus, three more independent The expression of the PPARγ gene was assessed at 3, 7, and 14 days (d) from the beginning of the protocol. Data obtained by quantitative Real-Time PCR (qPCR) were normalized using three reference genes (TATA Binding Protein-TBP, Glyceraldehyde-3-Phosphate Dehydrogenase-GAPDH, Hypoxanthine Phosphoribosyltransferase 1-HPRT1); the normalized expression value of CTR at 3 days (black column 3d) was set up to 1, and all other gene expression values were reported to that sample. Data were reported as normalized fold change ± standard error of the mean (SEM); n = 3; * p < 0.05 vs. CTR 3d.

Effects of Oxytocin on the hASC Osteogenic Commitment
The effects of OXT on hASC osteogenic commitment were analyzed by culturing cells with OXT in the induction medium for the whole duration of the differentiation protocol. Osteogenesis was monitored by evaluating the amount of the Alizarin Red S dye, the mRNA expression of the osteogenic gene marker bone gamma-carboxyglutamate protein (BGLAP, also named osteocalcin) as well as the protein expression of the RUNX2, osteopontin (OPN), type 1 collagen and fibronectin.
In the first experiment, four OXT concentrations (30, 100, 500, and 1000 nM) were used during the entire osteogenic protocol. The Alizarin Red S staining, which specifically binds calcium deposits, revealed a dose-dependent increase in the osteogenic commitment in cells cultured in an osteogenic medium (OM) and treated with OXT, especially at the concentration of 1000 nM (Figure 7a). Thus, three more independent experiments were performed using only the 1000 nM OXT concentration. Images and Alizarin Red S staining quantification data showed that OXT treatment induced an increase in the osteogenic differentiation potential of hASCs cultured in OM compared to the control counterpart (hASCs cultured in osteogenic medium without OXT treatment) (Figure 7b,c). Osteogenesis was then evaluated by measuring the mRNA abundance of the BGLAP gene in hASCs cultured in OM and treated without or with 1000 nM OXT for 3, 7, 14, and 21 days from the beginning of the osteogenic induction protocol. The expression of BGLAP followed the same expression pattern in both CTR and OXT-treated cells, i.e., an increase and then a decrease in BGLAP expression over time, but in OXT-treated hASCs an increase in BGLAP expression was observed after 3 days from the beginning of the protocol compared to CTR (Figure 7d), suggesting that the OXT treated cells were prone to initiate osteogenesis earlier than the untreated ones.
Finally, osteogenesis was investigated by analyzing the expression of specific osteogenic proteins by immunofluorescence. First, protein expression of RUNX2 and OPN was evaluated in hASCs cultured in an osteogenic medium with or without 1000 nM OXT after 3, 7, 14, and 21 days from the beginning of the osteogenic protocol. Images and fluorescence quantifications showed that both RUNX2 and OPN were statistically more expressed in OXT-treated hASCs compared to CTR cells after 3 days from the start of osteogenic protocol (Figure 8), while they are comparable at 7, 14, and 21 days.
Immunofluorescence staining images and the relative fluorescence quantification revealed that the expression of type I collagen was evident from the third day of the osteogenic protocol and that both CTR and OXT-treated hASCs showed both intracellular and extracellular collagen at 14 and 21 days (Figure 9). Type I collagen increased in OXTtreated cells compared to untreated cells at days 7, 14, and 21 of the osteogenic process, reaching statistical significance both at 7 and 21 days (Figure 9).
Finally, fibronectin was more expressed in OXT-treated cells than in untreated cells from day 3 of the induction protocol for the entire period analyzed, significantly at days 3 and 14 ( Figure 10). experiments were performed using only the 1000 nM OXT concentration. Images and Alizarin Red S staining quantification data showed that OXT treatment induced an increase in the osteogenic differentiation potential of hASCs cultured in OM compared to the control counterpart (hASCs cultured in osteogenic medium without OXT treatment) (Figure 7b,c). Osteogenesis was then evaluated by measuring the mRNA abundance of the BGLAP gene in hASCs cultured in OM and treated without or with 1000 nM OXT for 3, 7, 14, and 21 days from the beginning of the osteogenic induction protocol. The expression of BGLAP followed the same expression pa ern in both CTR and OXT-treated cells, i.e., an increase and then a decrease in BGLAP expression over time, but in OXT-treated hASCs an increase in BGLAP expression was observed after 3 days from the beginning of the protocol compared to CTR (Figure 7d), suggesting that the OXT treated cells were prone to initiate osteogenesis earlier than the untreated ones.   Finally, since OXT modulated autophagy in hASCs after 72 h of treatment, we evaluated the expression of two autophagy marker genes (BECN1 and MAP1LC3A) along the entire osteogenic process. Cells were cultured in the osteogenic medium as previously described and treated with or without 1000 nM OXT. For each condition, RNA was extracted 3, 7, 14, and 21 days after the start of treatment. Both genes significantly increased their expression in OXT-treated cells on day 3 of the induction protocol compared to CTR cells, while they showed the same expression pattern on days 7, 14, and 21 ( Figure 11).
Finally, osteogenesis was investigated by analyzing the expression of specific osteogenic proteins by immunofluorescence. First, protein expression of RUNX2 and OPN was evaluated in hASCs cultured in an osteogenic medium with or without 1000 nM OXT after 3, 7, 14, and 21 days from the beginning of the osteogenic protocol. Images and fluorescence quantifications showed that both RUNX2 and OPN were statistically more expressed in OXT-treated hASCs compared to CTR cells after 3 days from the start of osteogenic protocol (Figure 8), while they are comparable at 7, 14, and 21 days.  Immunofluorescence staining images and the relative fluorescence quantification revealed that the expression of type I collagen was evident from the third day of the osteogenic protocol and that both CTR and OXT-treated hASCs showed both intracellular and extracellular collagen at 14 and 21 days (Figure 9). Type I collagen increased in OXTtreated cells compared to untreated cells at days 7, 14, and 21 of the osteogenic process, reaching statistical significance both at 7 and 21 days (Figure 9).   Finally, fibronectin was more expressed in OXT-treated cells than in untreated cells from day 3 of the induction protocol for the entire period analyzed, significantly at days 3 and 14 ( Figure 10).   Finally, since OXT modulated autophagy in hASCs after evaluated the expression of two autophagy marker genes (BECN1 the entire osteogenic process. Cells were cultured in the osteogeni described and treated with or without 1000 nM OXT. For eac extracted 3, 7, 14, and 21 days after the start of treatment. B increased their expression in OXT-treated cells on day 3 of compared to CTR cells, while they showed the same expression pa 21 ( Figure 11).

Discussion
The aim of this study was to understand whether OXT could affect the main biological proprieties of hASCs, such as morphology, proliferation, viability, and senescence, as well as their differentiation potential. hASCs, like other MSCs, show Figure 11. Gene expression of autophagic markers along the osteogenic program in human adiposederived stem cells (hASCs) treated with Oxytocin (OXT). hASCs were cultured with or without (CTR) OXT 1000 nM. (a) Expression of Beclin 1 (BECN1) and (b) microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A) genes in hASCs CTR or treated with 1000 nM OXT was analyzed after 3, 7, 14, and 21 days (d) from the beginning of the osteogenic differentiation protocol. Data obtained with quantitative Real-Time PCR (qPCR) were normalized using three reference genes (TATA Binding Protein-TBP, Glyceraldehyde-3-Phosphate Dehydrogenase-GAPDH, and Hypoxanthine Phosphoribosyltransferase 1-HPRT1). In each graph, the normalized expression value of CTR hASCs at 3 days (black column 3d) was set up to 1, and all other gene expression values were reported to that sample. Data are reported as normalized fold change ± standard error of the mean (SEM); n = 3; * p < 0.05 vs. CTR 3d.

Discussion
The aim of this study was to understand whether OXT could affect the main biological proprieties of hASCs, such as morphology, proliferation, viability, and senescence, as well as their differentiation potential. hASCs, like other MSCs, show peculiar features that make them extremely attractive actors in the regenerative physiological processes and intriguing candidates for cell therapy approaches in the wider field of regenerative medicine.
Our data demonstrated that OXT did not influence hASC morphology at all OXT concentrations used and enhanced the expression of OXTR, which was expressed even at the basal level in hASCs. Our data are consistent with previously published data showing that OXTR was expressed in adipocytes [7] as well as in mouse ASCs [31] or hASCs [32]. Our observation that OXT enhanced the transcription of its own receptor coding gene suggests that OXT itself may be engaged in tonic feedforward mechanisms sustaining its action in an autocrine/paracrine fashion.
Moreover, under our experimental conditions, OXT did not affect cell viability, as well as cell proliferation, both at cellular and molecular levels. As a matter of fact, the expression of the genes involved in cell proliferation and cell cycle control MKI67, CCND1, CDKN2A, CDKN1A, and TP53 was not affected in OXT-treated cells, confirming that OXT did not influence cell proliferation. However, other researchers observed that OXT improved MSC proliferation, particularly in hPDLSCs and rat BM-MSCs [19,34]. This discrepancy could be related to the different cellular models as well as to different OXT concentrations which were used.
A coherent result was obtained from the migration assay where cells treated with OXT for 24 h showed no differences in the ability to close the scratch "wound" compared to CTR cells, even here in contrast with other studies where OXT promoted migration, although in different MSC models, as hUCB-MSCs, hPDLSCs, and rat BM-MSCs [20,34].
We then investigated the role of OXT in hASC senescence. Surprisingly, we found an increase in the number of positively blue-stained cells in hASCs treated with OXT. Since the induction of cellular senescence appeared to be in contrast with the general role of OXT mainly involved in health processes, we investigated the senescence pathway by analyzing the expression of cell senescence genes such as TERT and BMI-1: OXT did not alter the expression of both genes, suggesting that the increase on SA-β-Gal activity could not be related to the induction of a senescence process. In fact, it is known that the increase in SA-β-Gal activity is associated with other cellular processes, such as autophagy, in which an increase in lysosomal activity is required [37,38]. Therefore, we investigated the expression of two autophagic marker genes, BECN1 and MAP1LC3A, encoding proteins implicated in the autophagosome formation during the autophagy process [39,40]. The increase in the expression of both genes in hASCs treated with OXT indicated a possible involvement of OXT in the autophagy process, as already described in the mouse hepatocytes cell line AML12 [6], but never demonstrated in MSC models.
Finally, we analyzed the role of OXT on hASC differentiation ability, particularly focusing on both adipogenesis and osteogenesis. OXT was added to the adipogenic or osteogenic induction medium for the entire period of the differentiation protocol.
As demonstrated both by O.R.O staining and by the evaluation of PPARγ gene expression, lower concentrations of OXT did not influence the adipogenic process, which was instead reduced by the OXT's highest dose. These data confirmed the inclination of OXT to reduce the adipogenic potential already described by another research, but with a much lower dose of OXT [32].
On the other hand, osteogenesis was clearly promoted by OXT in a dose-dependent manner, and the most evident result was obtained with OXT at 1000 nM. Therefore, we studied the modulation of osteogenesis deeply throughout its process when OXT at 1000 nM was used on hASCs. The increase in red staining of Alizarin in OXT-treated cells was confirmed by the significant increase in BGLAP gene expression three days after the start of the osteogenic protocol; the pattern of BGLAP expression was similar in CTR and OXTtreated hASCs during the osteogenic protocol, but the evident increase in its expression at 3 days could suggest that OXT stimulated the cells to the osteogenic process, acting in the first phase of the differentiation, leading to an early expression of osteogenic markers compared to CTR. Akin to this view, two osteogenic marker proteins, RUNX2 and OPN, were statistically more expressed in OXT-treated hASCs than in untreated cells 3 days after initiation of the osteogenic protocol, strengthening the hypothesis of a push by OXT on hASCs towards osteogenic commitment in the first step of the process.
Autophagy is known to play a key role in maintaining stemness properties and modulating stem cell differentiation [41,42], including osteogenic differentiation of human MSCs [43,44]. Since OXT alone was able to induce the expression of BECN1 and MAP1LC3A, we hypothesized that the potentiation of osteogenic differentiation in OXTtreated hASCs may be due to OXT's ability to modulate the autophagy process. The data seem to support our hypothesis; in fact, the gene expression of BECN1 and MAP1LC3A increased significantly in OXT-treated hASCs at day 3 of osteogenic induction, as occurred with the osteogenic marker genes and proteins studied. This evidence suggests that OXT could promote osteogenesis through the potentiation of the autophagic process. Moreover, it is known that autophagy is closely linked to lysosomal activity, which is enhanced during osteogenic differentiation [45]. Thus, the increase in SA-β-Gal activity in OXT-treated cells might reinforce the hypothesis that OXT-induced osteogenic differentiation regulating autophagy and the lysosomal function.
In addition, type 1 collagen and fibronectin were analyzed along the osteogenic protocol. Type 1 collagen is an important protein of the bone extracellular matrix (ECM), where it binds to other proteins and cell surface integrins. Although present in many other cell types, type 1 collagen mediates cell differentiation of the osteoblast phenotype, and its mRNA level generally increases 2 days after osteogenesis induction [46,47]: for this reason, it is considered an early marker of osteo-differentiation. In fact, its expression was evident both in OXT-treated and untreated cells after 3 days from the beginning of the osteogenesis process, and appeared more pronounced in OXT-treated hASCs compared to CTR cells at 7, 14, and 21 days. A similar expression pattern was observed for fibronectin, known to be involved in the early stages of osteogenesis [48].
Together these results have shown that OXT promotes osteogenesis and reduces adipogenesis, confirming data already published, albeit in other experimental conditions [32,34].
These findings may be particularly rewarding. In fact, it is increasingly becoming evident that the proneness of suffering a bone fracture is not solely resulting from a decrease in bone mineral density [49]. Rather, preventing the fracturing risk within the context of osteoporosis appears to require the preservation of peculiar cues within the bone ECM. To this end, the maintenance of a proper composition in type I collagen and fibronectin will strongly contribute to fracture resistance beyond mineral bone strength. To this end, it is also worthy of considering the emerging role of fibronectin, an evolutionarily conserved glycoprotein detectable in all tissues, in promoting bone fracture healing, acting as a threedimensional scaffold immediately after trauma, orchestrating the assembly of multiple ECM components [50]. The currently observed capability of OXT to couple an increase in the osteogenic process with an enhanced expression of key players in ECM-mediated tissue strength may set the basis for deploying the use of OXT in a novel landscape of bone and fracture toughness. The potential biomedical implication of our observations is further supported by the compelling evidence for a remarkable excess of mortality risk in both women and men following an osteoporosis-related hip fracture [51], as well as the persistence of mortality excess in a wide variety of non-hip fragility fractures [52].

Materials and Methods
4.1. hASCs: Harvesting, Culture, and Characterization hASCs were isolated from the adipose tissue of 3 healthy female subjects, as previously described [36], and this study was approved by the Institutional Ethics Com- Thermo-Fisher Scientific, Waltham, MA, USA) and were maintained in standard culture conditions at 37 • C with 5% carbon dioxide (CO 2 ) in a humidified atmosphere. The medium was replaced after 4 days from the seeding and then twice a week until the confluence. When reaching 80% of confluence, hASCs were passaged by treatment with trypsin-EDTA (Sigma-Aldrich Co., St. Louis, MO, USA). Experiments were performed using hASCs at the 3rd-6th culture passage. The hASC characterization and their trilineage differential potential was assessed as described in our previous manuscript [36].

Morphological Assessment and OXTR Gene Expression
To analyze hASC morphology, cells were seeded in a 24-well plate (Corning Incorporated, Corning, NY, USA) at a density of 3500 cells/cm 2 . After 24 h in standard conditions, cells were exposed for 72 h to different final concentrations of OXT (100, 500, and 1000 nM). At the end of the exposure time, cells were observed under a light microscope Leica Labovert FS inverted Microscope (Leica Microsystems, Wetzlar, Germany), and images were acquired with a Leica MC170 HD Imaging System Camera (Leica Microsystems, Wetzlar, Germany).
To evaluate the expression of the OXTR, hASCs were seeded in 25 cm 2 flasks (Corning Incorporated, Corning, NY, USA) at a density of 3500 cells/cm 2 . After 24 h in standard conditions, cells were treated with OXT 1000 nM for 72 h, and then harvested for RNA isolation. Untreated cells were used as CTR. RNA was retrotranscribed in cDNA and used in qPCR analysis (see Section 4.10).

Cell Viability
hASCs were seeded in a 6-well plate (Corning Incorporated, Corning, NY, USA) at a density of 3500 cells/cm 2 . After 24 h in standard conditions, cells were treated with OXT (100, 500, and 1000 nM) for 72 h (2 wells/condition). Then, cells were detached by trypsin-EDTA and counted. Cells were resuspended in a solution containing 50% of Erythrosine B (Sigma-Aldrich Co., St. Louis, MO, USA) red dye 0.2% in Phosphate Buffered Saline (PBS, Corning Incorporated, Corning, NY, USA). Viable and dead cells (not stained and red stained, respectively) were manually counted (at least twice for each condition) using the Neubauer hemocytometer (BRAND GmbH, Wertheim, Germany) and a light microscope Leica Labovert FS inverted Microscope (Leica Microsystems, Wetzlar, Germany). For each sample, cell viability was obtained by calculating the percentage of living cells compared to the total number of cells.

BrdU Assay and Expression Analysis of Proliferation/Cell Cycle Markers
BrdU assay (Roche, Basel, Switzerland) was used to evaluate OXT effects on cell proliferation. hASCs were seeded in a 96-well plate (Corning Incorporated, Corning, NY, USA) at a density of 6000 cells/cm 2 (3 wells/condition). After 24 h in standard conditions, cells were treated with OXT (100, 500, and 1000 nM) for 72 h and then incubated with 20 µL/well BrdU labeling solution for 3 h at 37 • C, and then fixed with 200 µL/well FixDenat reagent for 30 min at room temperature (RT) (according to the BrdU manufacturer's instructions). A negative control (medium without cells) was included. Next, hASCs were incubated with peroxidase-conjugated anti-BrdU antibody (AB) and then with the peroxidase substrate. The reaction was interrupted by adding 25 µL of H 2 SO 4 1 M to each well. The absorbance at 450 nm was measured with the Wallac 1420 Victor2 Multilabel Counter (Perkin Elmer, Waltham, MA, USA). hASC proliferative ability was expressed as a percentage of BrdU incorporated in treated cells compared to CTR cells (set to 100%) ± standard deviation (SD).
To assess the expression of genes involved in proliferation (MKI67) and cell cycle control (CCND1, CDKN2A, CDKN1A, and TP53), hASCs were seeded in 25 cm 2 flasks at a density of 3500 cell/cm 2 . After 24 h in standard conditions, cells were treated with OXT 1000 nM for 72 h, and then harvested for RNA isolation (see Section 4.10). Untreated cells were used as CTR. RNA was retrotranscribed in cDNA and then used in qPCR analysis (see Section 4.10).

Scratch Wound Healing Assay
hASC migration capability was assessed by performing a scratch wound healing assay. hASCs were seeded in a 24-well plate at a density of 20,000 cells/cm 2 (3 wells/condition). After 24 h in standard conditions, cells were treated with OXT (100, 500, and 1000 nM) for 24 h. Then, a scratch was made using sterile plastic tips. Wells were washed with PBS to remove detached cells. Fresh culture medium with or without OXT was added to the wells. Scratched monolayers were monitored, and images were acquired using a light microscope (Leica Labovert FS inverted Microscope; Wetzlar, Germany) with a Leica MC170 HD digital camera (Leica Microsystems, Wetzlar, Germany) at regular interval times (0, 12, 24, 36 h) until their complete closure. Scratch areas were measured using the ImageJ software 1.53e (National Institute of Health, Bethesda, MD, USA) according to the method described by Venter and Niesler [53]. Briefly, images were converted to 8-bit (grayscale), the edges found (Process → Find Edges), and the images blurred (Process → Smooth: ×20 times). To correctly identify the wound area, the threshold was manually adjusted. The wound area was quantified by analyzing particles of size between 150 µm to infinity (Analyze → Analyze Particles [size: 150-infinity]). The results are presented as the mean of the percentage of wound closure normalized on the CTR ± standard error of the mean (SEM). The percentage of wound closure at t = 0 h was set to 0 for both treated and untreated cells.

Senescence-Associated β-Galactosidase Staining and Gene Expression Analysis of Senescence/Autophagy Markers
Cell staining was performed using a SA β-gal commercial kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer's instructions. Briefly, hASCs were seeded in a 24-well plate at a density of 3500 cells/cm 2 (2 wells/condition). After 24 h in standard conditions, cells were treated with OXT (100, 500, and 1000 nM) for 72 h. After the exposure time, cells were fixed and processed according to the manufacturer's instructions. The staining reaction was incubated overnight. The number of positive (blue) and negative (not colored) cells was counted in each condition in 5 random fields (10 fields/condition) under the microscope Leica Labovert FS inverted Microscope (Leica Microsystems, Wetzlar, Germany). Results are expressed as the percentage of SA β-gal-positive cells ± SD.
To study the expression of genes involved in senescence (BMI-1 and TERT) or in autophagic processes (BECN1 and MAP1LC3A), hASCs were seeded in 25 cm 2 flasks at a density of 3500 cell/cm 2 . After 24 h in standard conditions, cells were treated with OXT 1000 nM for 72 h, and then harvested for RNA isolation (see Section 4.10). Untreated cells were used as CTR. RNA was retrotranscribed in cDNA and then used in qPCR analysis (see Section 4.10).

Adipogenic Commitment: O.R.O Staining and PPARγ Expression Analysis
The adipogenic differentiation capability of hASCs was evaluated in the presence of OXT for 14 days. Cells were seeded in 48-well or in 6-well plates (Corning Incorporated, Corning, NY, USA) at a density of 9000 cells/cm 2 (2 wells/condition) for O.R.O staining and gene expression analysis, respectively. At 100% confluence, cells were cultured with standard medium (SM) or StemPro Adipogenesis Differentiation medium (Thermo-Fisher Scientific, Waltham, MA, USA) in the presence or absence of OXT. Untreated cells, both in SM and adipogenic medium, were used as control (CTR). Media were changed twice a week for 14 days.
The qualitative and quantitative assessment of adipogenic differentiation was evaluated in hASCs cultured in the absence or presence of OXT (30,  For the expression analysis of the PPARγ adipogenic marker, hASCs were treated only with OXT 1000 nM. RNA was extracted and retrotranscribed, as described in Section 4.10, at 3, 7, and 14 days from the beginning of the induction protocol. Finally, the obtained cDNA was used in qPCR analysis (see Section 4.10).

Osteogenic Commitment: Alizarin Red S Staining and Expression Analysis of Osteogenic/Autophagy Markers
Cells were seeded in 24-well plates or 6-well plates at density of 5000 cells/cm 2 (2 wells/condition) for Alizarin Red S staining and gene expression analysis, respectively. At 70% confluence, cells were cultured with standard medium (SM) or StemPro Osteogenesis Differentiation medium (OM) (Thermo-Fisher Scientific, Waltham, MA, USA) in the presence or absence of OXT. Untreated cells, both in SM and OM, were used as CTR. Media were changed twice a week for 21 days.
The hASC capability to differentiate toward the osteogenic lineage was assessed with a qualitative and quantitative evaluation of calcium deposits in the absence or presence of OXT (30, 100, 500, and 1000 nM) at the end of the differentiation protocol (21 days). Briefly, cells were fixed with 4% formaldehyde in PBS (Sigma-Aldrich Co., St. Louis, MO, USA) in a deionized water solution (pH = 4.1) for 15 min. The exceeding dye was removed by washing wells with deionized water. Images were acquired using the Leica Labovert FS Inverted Microscope (Leica Microsystems, Wetzlar, Germany) with a Leica MC170 HD Imaging System Camera (Leica Microsystems, Wetzlar, Germany). To quantify the number of calcium deposits, the Alizarin Red S dye was solubilized with 10% of acetic acid (Sigma-Aldrich Co., St. Louis, MO, USA); the solution was loaded in a 96-well plate in technical triplicate to measure the absorbance (Abs) at 405 nm using a spectrophotometer plate reader (Wallac 1420 Victor2 Multilabel Counter; Perkin Elmer, Waltham, MA, USA). Data are reported as the mean of the Abs ± SD.
For the expression analysis of BGLAP, BECN1, and MAP1LC3A (osteogenic and autophagy markers, respectively), hASCs were treated only with OXT 1000 nM. RNA was extracted and retrotranscribed, as described in Section 4.10, at 3, 7, 14, and 21 days after the beginning of the induction protocol. Finally, the obtained cDNA was used in qPCR analysis (see Section 4.10).

Conclusions
In recent years, the importance of hASCs in the promising field of cell therapy has increasingly emerged, in particular for their differentiative capacity, such as that towards osteogenic commitment. In the present study, we have shown that OXT, which we already know plays an important role in health and well-being, is able to promote hASCs towards an osteogenic commitment, acting as a driving force in the early days of the differentiation pathway. In association with this, we observed how OXT is also able to activate autophagy marker genes in the same period of time. The capability of OXT to elicit a significant increase in type I collagen and fibronectin, together with an increase in osteogenesis and autophagy, may set the basis for novel bone and fracture toughness approaches beyond those devoted mainly to promoting bone strength via an increase in bone mineral density. Further in vitro and in vivo studies will be needed to address this wide area of inquiry.