Effect of mesenchymal stem cells combined with chondroitin sulfate in an osteoarthritis in vitro model

Introduction: Osteoarthritis (OA) is a degenerative joint disease which affects the whole joint structure. Many authors have focused on the factors responsible for the development of inammatory processes involved in OA. Adipose tissue-derived mesenchymal stem cells (ASCs) represent a promising alternative of cell-based therapy strategy in the treatment of OA which could be combined with any other drug. Chondroitin sulfate plays a protective role in the joint based on the decrease of pro-inammatory cytokines, thus having an important role in activating and inhibiting the metabolic pathways in chondrocytes. Aims: The effectiveness of chondroitin sulfate and ASCs combined in an in vitro model of OA has been evaluated in this study. Materials: Cytokines and factors which are involved in OA as well as specic cartilage gene expression after adding ASCs and chondroitin sulfate have been discussed in detail. Results: Our results show a decrease in the expression of all genes related to the pro-inammatory cytokines analysed. Although there was no increase in the expression of the specic genes of the cartilage matrix, such as collagen type II and aggrecan. Conclusions: This study shows the effectiveness of association of ASCs and chondroitin sulfate for the treatment of OA.


Introduction
Osteoarthritis (OA) is considered to be the fourth leading cause of disability by the year 2020 [1]. OA is a destructive joint disease causing degeneration of cartilage [2,3]. Currently, in ammation is considered to be included in the development and progression of OA in the early stages [4]. Emerging experimental evidence shows that secreted in ammatory factors such as pro-in ammatory cytokines are critical mediators of the disturbed metabolism and enhanced OA catabolism [5]. These cytokines mediate cartilage destruction through the upregulation of in ammatory or catabolic genes and the downregulation of anti-in ammatory or anabolic genes in articular chondrocytes [6]. In particular, IL-1 reduces the expression of type II collagen (Col2A1) [7] and increases the production of matrix metalloproteinases (MMPs) [8,9], prostaglandin E 2 (PGE 2 ), cytokines, chemokines, reactive oxygen species, and nitric oxide (NO) [10,11]. These substances enhance the catabolic activity of the chondrocytes and cause the destruction of the cartilage matrix.
Chondroitin sulfate (CS) is a major component in the extracellular matrix in many connective tissues [12,13]. Commonly referred to "symptomatic slow-acting drug in OA" (SySADOA), CS is extensively used in the management of OA patients [14]. In vitro, CS has been shown to have anti-in ammatory and anticatabolic properties on chondrocytes [15] and to be a structure/disease modifying anti-osteoarthritis drug (S/DMOAD) [16][17][18].
Cellular therapies for treating early to late stage OA have been thoroughly researched into for over two decades. Tissue engineering using stem cells emerged as an alternative method for treating OA over the last 10 years [19,20]. In this way, mesenchymal stem or stromal cells (MSCs) have been extensively researched into mainly for their regenerative potential [21][22][23]. Interestingly enough, stem cells are capable of secreting a wide range of trophic mediators which can exert paracrine effects on other cell types. Thus adipose tissue-derived MSCs (ASCs) are an interesting alternative MSCs source (instead of bone marrow stem cells, BMSCs) which can be easily collected using liposuction [24,25].
The injected or infused MSCs are based on two activities: immunomodulation and trophic activities. The immunomodulation of these cells has been shown to be mediated by both secreted bioactive molecules and by cell-cell contact and could involve suppression of T-cell proliferation in response to alloantigens or mitogens, inhibition of B cell proliferation, as well as dendritic cell maturation, and promoting the generation of T-regulatory cells [26]. Some of these cytokines and factors are Nuclear factor kappa B (NF κB), transforming growth factor beta (TGF-β), indolamine 2,3-dioxygenase (IDO), interleukin 6 (IL-6) which are involved in MSCs immunomodulation [27] as well as others involved in the renewal of the extracellular matrix, such as type II collagen [28,29].
The aim of this work is to study the effect of ASCs and CS on in ammatory mediators and proteolytic enzymes induced by TNF and related to cartilage catabolism.

Ethical Disclosure
The authors state that the experimental procedures carried out in this work were approved by the Medical Committee of the University Hospital of León. Written consent was obtained from all patients following to the Helsinki Declaration of 1975, as revised in 2008.

Materials
In this study, human cells were obtained from three patients with OA symptoms (n= 3 donors; a 74-yearsold male and two females aged 67 and 55). Chondrocytes were obtained from femoral cartilage. ASCs were obtained from Hoffa adipose tissue. As controls healthy chondrocytes from Innoprot ® (Bizcaia, Spain) were used.

Isolation and Culture of ASCs and Chondrocytes
ASCs were isolated from adipose tissue obtained from the knee fat deposit region, also known as the Hoffa fat pad [30]. Cells were collected and plated in 25cm 2 culture asks (IWAKI ® , Japan, Code: 3100-025).

ASCs differentiation
Isolated ASCs were cultured under conditions conducive to adipogenesis, osteogenesis and chondrogenesis to be assessed for multi-potentiality. After 15 days the cultured cells in adipogenic medium were stained with Oil Red O. ASCs were cultured under osteogenic culture conditions for 15 days.
After induction, con rmation of osteogenesis was achieved by alizarin red staining. Finally, cell cultures were incubated under chondrogenic medium. Three weeks after induction con rmation of chondrogenesis was achieved through alcian blue staining and confocal microscopy using anti-Col2a1 (Abcam ® , UK, Code: 185430) (1:100).

Fluorescence microscope proliferation assay
Density of 1x10 6 ASCs and chondrocytes were labeled with 5μM of CellTrace ® Violet proliferation tracking dye and CellTrace ® CFSE dye (Green) (Invitrogen ® , USA, Codes: C34554, C34554 ). Cell proliferation was analyzed at 24 and 36h. Two-dimensional images were digitally recorded for each surface at sample center as single topographical location with Nikon Eclipse TE2000-U inverted microscope (Nikon ® , Japan).
Flow cytometry proliferation assay Relative quanti cation was carried out normalizing to the housekeeping gene ACT-β. Primers were designed using an OLIGO7 ® primer design tool (

ELISA
Chondrocytes and ASCs were stimulated with TNF (10µg/mL) for 12h. Concentration of prostaglandin E 2 (PGE 2 ) was measured by a speci c ELISA with a goat anti-Mouse IgG microtiter plate, following the manufacturing instructions (Enzo Life Sciences Inc ® , USA). Measurements were carried out using a reader set Multiskan ® GO Microplate Spectrophotometer (Thermo Fisher Scienti c ® , USA) at 450 nm.
Concentrations were calculated by comparing them to known standards.

Statistical Analysis
Each result from the study was expressed as the mean ± SD and was carried out with 3 experimental replicates. Statistical analysis was carried out using IBM ® SPSS ® Statistics (USA). Signi cant differences among the groups were determined using ANOVA followed by post-hoc analysis for multiple group comparisons or Student t-test for two group comparisons. Results with p<0.05 were considered statistically signi cant.

ASCs characterization
ASCs from infrapatellar Hoffa fat were characterized at passages 2 and 3. ASCs are adherent cells of broblastic morphology. With regard to cell pattern, it took 7-9 days to reach con uence in T75 culture ask, once they had been extracted.
Flow cytometric characterization showed a high expression of CD105 (endoglin), CD73 (ecto-5'nucleotidase) and CD90 (Thy1). The uorescence microscopy analysis reveals that cells displayed positive results for the surface markers CD90, CD73 and CD 105, as was expected. The percentages of positive cell markers and their histograms are shown in Figure 1 A-C.
The tri-lineage differentiation potential of the ASCs was con rmed With regard to ASCs differentiation, after 15 days cells had differentiated towards osteocytes, chondrocytes and adipocytes. Spherical nodules were observed in chondrogenic cultures and deposits of acid mucopolysaccharides were con rmed with alcian blue staining. Similarly, under osteogenic induction conditions, dark ECM deposits were detected after the induction period and mineralized calcium deposits stained orange-red with alizarin red S. ASCs, which underwent adipogenic differentiation, were characterized by an accumulation of cytoplasmic triglycerides and were represented as lipid droplets using Oil Red O staining (Fig. 1 D-G).

Cell proliferation in ASCs and chondrocytes co-cultures
In order to study the behavior of ASCs and chondrocytes in co-cultures, a proliferation analysis was also carried out using uorescence microscopy and ow cytometry. Cell proliferation was analyzed at 12, 24 and 36 hours with or without TNF and CS depending on the condition involved. ASCs were stained in violet and chondrocytes in green. The results obtained using confocal microscopy and ow cytometry are shown in Figure 2. Under all experimental conditions chondrocytes proliferation rates were higher than ASCs. The proliferation rate was not the same between either cell lines due to the larger size of ASCs.
The addition of 10 µg/mL of in ammatory agent (TNF) slightly reduces the viability of chondrocytes, which stablizes when CS is added. The addition of CS does not signi cantly affect the proliferation of any cell types since the cell viability remains stable (Fig. 2C).

Effect of ASCs and CS on NF-κB translocation
It is known that NF-κB is involved in the effects of TNF on in ammatory and catabolic mediators, in this key transcription factor. We studied the possible regulation of ASCs and CS. TNF quickly induced translocation of NF-κB into the nucleus and binding of DNA to trigger off transcription of genes. In Figure  3 is shown the TNF-induced p65-NFκB binding of DNA in in amed chondrocytes. TNF rapidly initiates translocation of NF-κB into the cell nucleus and authoritative translation of DNA to enact consistency. When ASCs were in amed a decreasing in the DNA binding of p65-NFκB was observed. In Figure 4 an improvement in the restriction of p65-NFkB DNA in excited chondrocytes incited by TNF is shown. CS and ASCs has substantially diminished the binding of P65-NFκB to DNA.
CS is capable to reducing the concentration of PGE 2 in previously in amed cells We observed the effect of CS when PGE 2 was released under different experimental conditions using ELISA. The dose of CS (200 ng/mL) used was the recommended by other authors [31] and was the dose with which the best results were obtained in previous experiments carried out in our lab (results not shown). In cells which did not receive an in ammatory stimulus, PGE 2 concentration remained at very low basal levels. However, in TNF-stimulated cells the level of PGE 2 production increased considerably (Fig. 5).
In a detailed analysis, our data showed that PGE 2 production only decreased when chondrocytes were cocultured with ASCs, and CS was added. The addition of CS into stimulated chondrocytes co-cultured with ASCs signi cantly reduces PGE 2 production when it is compared with the same co-culture without CS.
Thus, the promising treatment with a combination of ASCs and the CS causes the greatest decrease in prostaglandin E 2 concentration, and an abatement in its release of 28.26% was observed. IL-6, iNOS, TNF, MMP-13, IDO and TGF-β expression in ASCs-chondrocytes co-cultures We analyzed the expression of IL-6, iNOS, TNF and MMP-13 in co-cultures of choncrocytes and ASCs with CS added. In the different experimental conditions in ammation was induced with TNF. Results are given in Figure 6. The expression of IL-6 and iNOS signi cantly increased (p≤ 0.005) in chondrocytes cultures stimulated with TNF. However, when CS was added IL-6 expression signi cantly reduced. IL-6 expression increased in ASCs cultures when TNF was added but less than in chondrocyte cultures. Nonetheless, its expression reduced when chondrocytes were co-cultured with ASCs and treated with CS.
When iNOS expression was examined in chondrocytes after stimulation with TNF, it was found that chondrocytes showed extremely high iNOS levels. The treatment of chondrocytes with CS and ASCs dramatically reduced iNOS gene expression (Fig. 6). When CS was added to ASCs and chondrocytes cocultures stimulated with TNF, iNOS expression was lower, though not signi cantly lower.
With regard to TNF and MMP-13 the same effect was observed in both ASCs and chondrocytes in response to in ammatory cytokine exposure, which reduced when treatment with CS occurred (Fig. 6), reaching the same levels as non-stimulated cells.
TGF-β levels increased slightly when activated with TNF (for ASCs and chondrocytes) but values were very close to those in the chondrocytes control group. In ASCs-chondrocytes co-cultures stimulated with TNF and when CS was added TGF-β levels were reduced signi cantly (Fig. 6).
IDO was over-expressed in stimulated chondrocytes and ASCs, but also when the treatment was initiated with CS its expression increased but not signi cantly with very different values as given in the error bar in Figure 6. Regarding this gene, no reliable conclusions can be extracted with respect to this gene.

Speci c Chondrogenic Gene expression evolution
The expression of speci c chondrogenic genes such as SOX-9, aggrecan (ACAN) and collagen type II (Col2a1) was analyzed under the different experimental conditions stimulated and non-stimulated with TNF (Fig. 7). We observed that levels of Col2a1 and ACAN increased signi cantly when CS was added to chondrocytes cultures without TNF. However, the expression of the three speci c genes of chondrogenesis did not signi cantly change when TNF and CS were added. The same effect was detected in the co-cultures where an increase in the gene expression of Col2a1; ACAN and SOX-9 was obtained, though not signi cantly.

Discussion
MSCs are an attractive alternative candidate to the conventional treatment for regenerative therapies [32]. They have been suggested as a new cell source for OA treatment because of their capability of differentiating in chondrocytes and the paracrine effects of secreted bioactive substances as well as their immunomodulatory effects [33]. CS is the major GAG component of native cartilage tissue which could provide cues to stimulate cells so as to proliferate, migrate, differentiate and produce the ECM compounds [34]. CS has also been shown to have anti-in ammatory effects reducing the concentration of pro-in ammatory cytokines such as TNF [35,36] and IL-1β [37]. We hypothesized that the combination of ASCs with CS should be capable of enhancing the cartilage regeneration and diminishing the in ammation in an in vitro model of OA. We evaluated the immunomodulatory effect of CS combined with ASCs in co-culture with in amed chondrocytes. The expression of speci c cartilage genes was also analysed.
Most previous studies which examined chondrocyte depletion during OA progression discovered that a variety of factors (including TNF) have been reported to induce a progressive cartilage joint degeneration in OA [38,39]. In a proliferation assay we observed that the addition of in ammatory cytokine (TNF) slightly reduces the viability of chondrocytes, which stabilizes when CS is added. The addition of CS does not signi cantly affect the proliferation of any cell types since the cell viability remains very stable.
It has been reported that MSCs could be induced to express enhanced levels of IDO and PGE 2 . ASCs are known to constitutively produce PGE 2 and this production signi cantly increases in co-cultures [40][41][42].
However, in our work PGE 2 concentration remained at very low base levels in cells which did not receive an in ammatory stimulus. According to other references, CS had no effect on the basal PGE 2 release [43].
The effect of TNF and IL-1, both in chondrocytes [44] and in MSCs [40] causes the increase of PGE 2 expression. Although PGE 2 is considered a pro-in ammatory cytokine, there exist different theories on the bene cial or detrimental effect which it produces on OA [45]. Ronca et al., 1998 [46], showed that the effects of CS in the treatment of OA are due to various mechanisms of action which decrease the concentration of PGE 2 in the joint c [47,48]. However, we observed that in TNF-stimulated cells the level of PGE 2 production increased considerably under all conditions. The amount of PGE 2 produced was signi cantly decreased, although it remained high compared to the non-stimulated controls under conditions of co-culture with CS and TNF in the culture medium. Currently, PGE 2 seem to be involved in the up-regulation of the anti-in ammatory cytokine interleukin (IL)10 while reducing the secretion of TNF [49,50]. We can therefore speculate that the inhibition of PGE 2 production by CS could reduce the degradative effect in the OA deep zone cartilage. Lastly, CS is shown to inhibit the expression of enzymes involved in PGE 2 synthesis, COX-2 and mPGES-1 [51].
As will be discussed later on, ASCs reduce levels of certain pro-in ammatory cytokines whose production is associated with PGE 2 . Therefore, CS and ASCs jointly manage to reduce OA processes through several different routes. Our results agree with those obtained by other authors. Cytokine IL-6 is responsible for pain in OA [51], being one of the main inducers of in ammation. MSCs reduced the levels of proin ammatory cytokines, including IL-6 [43]. With regard to PGE 2 analysis, it is known that it accelerates expression of pain-associated molecules such as IL-6 e iNOS [51], but MSC-derived PGE 2 always acts independently of IL-6 [41]. In our work, we observed that the expression of IL-6 signi cantly decreased in co-culture of ASCs and chondrocytes previously in amed and with the addition of CS, in comparison with the in amed chondrocytes without CS. Previous studies showed these effects in which MSCs and CS reduced the expression of pro-in ammatory cytokines, among which were IL-6 [52]. The combination of ASCs and CS resulted in a marked reduction in the expression of IL-6, although it was not as important as that produced solely by CS. Although it has been shown that IL-6 is one of the main interleukins which induce in ammation, its role is currently being debated due to evidence that this interleukin could have an anti-in ammatory role [53].
Contrary to what happened in the previous case for IL-6, the expression of iNOS underwent a greater reduction in the cultures of ASCs combined with CS as a treatment. Both CS and ASCs signi cantly reduced their expression, this reduction was more than 40 times lower with the use of CS and up to 50 times less when CS and ASCs were combined. When iNOS expression was examined in chondrocytes after stimulation with TNF, it was found that chondrocytes expressed extremely high iNOS levels, which agree with Charles et al., 1993 [54]. However, the expression level in ASCs was minimal, as predicted by Ren et al., 2009 [55, 56]. As in the previous cases, the treatment of chondrocytes with CS and ASCs dramatically reduced iNOS gene expression.
With respect to metalloproteinase, activated chondrocytes also produced MMP-1 and MMP-13. Increased expression of IL-6 is related to the production of enzymes from the MMPs group [49]. Nevertheless, their levels were reduced in in amed chondrocytes treated with CS and ASCs, particularly MMP-13 production.
Deletion of the MMP-13 gene attenuated articular cartilage degradation (it targets: type II collagen), and it has been shown that it is a critical downstream target gene of TGF-β signaling during OA development [57]. It has recently been shown that global MMP-13 knockout could prevent articular cartilage erosion [58].
Both ASCs and in amed chondrocytes showed high TNF expression, which decreased when treatment with CS occurred. Its levels may not have been as high as expected since PGE 2 prevents proliferation of TNF [27]. This molecule together with IL-1β is considered to be a key in ammatory cytokine involved in the pathophysiological processes occurring in OA, and it affects blocking the chondrocytes synthesis of proteoglycan components, and Col2a1. Moreover, it is responsible for the increased production of iNOS and IL-6 [49].
TGF-β levels slightly increased when ASCs and chondrocytes were activated with TNF, although values were very close to those from the chondrocytes control group. In the presence of CS, levels were minimally reduced. Observations by Shen et al., 2014 [57]. have shown that TGF-β inhibition signaling in chondrocytes leads to chondrocyte terminal differentiation and development of OA, as this cytokine is responsible for stimulating the production of proteoglycans, Col2a1 and chondrogenesis. Other publications also con rm that the amount of TGF-β is low or even undetectable in patients with OA [49]. Lee et al., 2014 [42], have shown that the expression of IDO was induced in MSCs after tissue damage. Accordingly, in other tissues damaged or stimulated with TNF, the production of IDO increased [27]. This agrees with our results, which, after inducing cell in ammation, increased the expression of IDO, although it also increased with the addition of CS and ASCs. Our results showed that IDO is probably expressed as a natural protector against in ammation as shown by other authors [58].
During the more advanced stages of OA the amount of Col2a1 and ACAN decreases by denaturation [59,60], which was in uenced by the increase in the expression of MMPs. Levels of type II collagen and aggrecan increased signi cantly when CS was added to chondrocytes in in amed co-cultures. This raises questions as to the connection of Col2a1 and ACAN to the increase of proteoglycans thanks to the CS, key components of ECM. CS is widely distributed in matrix where it forms an essential component of proteoglycans by covalent links with proteins [43], aiding the regenerative process. Our results con rm the boosting effect of CS on some gene expression of cartilage extracellular matrix (ACAN, Col2a1). Moreover, the expression of these genes was always higher when the ASCs were present compared to the in amed chondrocytes without CS. There were no signi cant differences in SOX9 gene expression, a critical factor for chondrocyte differentiation that facilitates the expression Col2a1 [61].
Canonical NFκB activation, a nuclear translocation factor closely related to the initiation of the in ammatory cascade, plays a key role in chondrocytes in expressing MMPs, ADAMTS and in ammatory cytokines [62] themselves. MMPs expression in chondrocytes has been shown to be induced by TNF through pathways to activate mitogen-activated protein kinase (MAPK), kappa nuclear factor B (NF-kB), and protein activator 1 (AP-1) [63]. In order to understand the possible mechanism by which they are able to downregulate these mediators, we studied the effect of ASCs and CS on NF-κB translocation. Our ndings showed that the inhibitory effects of ASCs and CS on the expression of catabolic and proin ammatory molecules observed could be related to the reduction of NF-κB translocation in TNFin amed chondrocytes. Jomphe et al. [64], in an in vitro study has shown that CS inhibited the translocation of NFkB. The therapeutic e cacy of CS and ASCs may be due to their anti-in ammatory activity and the stimulation of proteoglycan synthesis, as well as the decrease in the catabolic activity of chondrocytes, inhibiting some proteolytic enzymes such as metalloproteases and in ammatory mediators (TNF, iNOS, IL-6, PGE 2 , NFkB) [18].

Conclusions
To sum up, we can conclude that CS in combination with ASCs provides very encouraging results in OA treatment by means of inhibition the synthesis of pro-in ammatory and degradative mediators known to exert a deleterious effect on the cartilage. The therapeutic bene t of CS and ASCs could be produced by at least three mechanisms that can contribute to delaying the progression of osteoarthritis: inhibition of the synthesis of in ammatory mediators (TNF, IL-6, PGE 2 and NO) mediated by TNF and interleukin-1, inhibition of the synthesis of catabolic enzymes such as MMP-13 and stimulation of the synthesis of extracellular matrix components such as collagen type II and aggrecan. Further research is needed regarding the molecular pathways which control the functional behaviour of cartilage under both physiological and pathological conditions to develop more effective strategies for the treatment of OA and other cartilage-related diseases.

Declarations Author Contributions
All the authors were involved in drafting the article or revising it critically for major intellectual content, and all the authors approved the nal version to be published. Dr. Villar-Suárez has had full access to all the data in this study and is responsible for the integrity of the data and the accuracy of the data   Effect of TNF on NF-κB activation in ASCs and chondrocytes. Cells were stimulated with TNF for 12 h and p65-NF-κB binding to DNA was determined by immuno uorescence.

Figure 4
Effect of ASCs and CS on NF-κB activation in in amed chondrocytes. Cells were stimulated with TNF for 12 h and p65-NF-κB binding to DNA was determined by immuno uorescence.

Figure 6
Relative expression of IL-6, iNOS, MMP-13, IDO, TNF and TGF-β in chondrocytes, ASCs and co-cultures of ASCs and chondrocytes. cell culture. CS was added to the cultures for 12 h and then in ammation was induced using TNF for 12 h. +++P ≤0.005) with regard to chondrocytes stimulated with TNF. # (P≤0.05) ### (P ≤ 0.005) with regard to chondrocytes non-stimulated with TNF.

Figure 7
Relative gene expression of speci c genes of chondrogenesis SOX-9, ACAN and Col2a1 in human chondrocytes, ASCs and co-culture of ASCs-Chondrocytes. CS was added to the cultures for 12 h and then in ammation was induced using TNF for 12 h. ***P ≤ 0.005 with regard to chondrocytes non stimulated with TNF.