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EUR CELL Mater: True Open Access ecmjournal.org
Sodium Hyaluronate supplemented culture media combined
with joint-simulating mechanical loading improves
chondrogenic differentiation of human Mesenchymal Stem
cells
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Journal: eCM Journal
Manuscript ID eCM-Oct-2020-OA-0091.R1
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Manuscript Type: Original Article
Date Submitted by the
30-Mar-2021
Author:
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Complete List of Authors: Monaco, Graziana; Research Institute Davos; Keele University
El Haj, Alicia Jennifer; University of Birmingham
Stoddart, Martin; Research Institute Davos; Keele University
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Alini, Mauro; AO Research Institute Davos, Musculoskeletal Regeneration
Hyaluronic acid, Mesenchymal Stem cells, chondrogenic differentiation,
Keywords: articular cartilage, hypertrophy, joint simulating bioreactor, mechanical
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loading, TGF-β1
In-vitro models aim to recapitulate the in vivo situation. To more closely
mimic the knee joint environment, current in vitro models need
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improvements to reflect the complexity of the native tissue. High
molecular weight hyaluronan (hMwHA) is one of the most abundant
bioactive macromolecules in healthy synovial fluid, while shear and
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dynamic compression are two joint-relevant mechanical forces.
The present study aimed to investigate the concomitant effect of joint-
simulating mechanical loading (JSML) and hMwHA supplemented-culture
media on the chondrogenic differentiation of primary human bone
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marrow-derived mesenchymal stem cell (hBMMSC).
hBMSC chondrogenesis was investigated over 28 days at the gene
expression level and total DNA, sulphated glycosaminoglycan, TGF-β1
production, and Safranin O staining were evaluated.
Abstract:
The concomitant effect of hMwHA culture media and JSML, significantly
increased cartilage-like matrix deposition and sulphated
glycosaminoglycan synthesis, especially during early chondrogenesis. A
stabilization of the hBMSC-derived chondrocyte phenotype was observed
through the reduced upregulation of the hypertrophic marker collagen X
and an increase in the chondrogenic collagen type II/X ratio.
A combination of JSML and hMwt HA media, better reflects the
complexity of the in vivo synovial joint environment. Thus, JSML and
hMwt HA media will be two important features for joint-related culture
models to more accurately predict the in vivo outcome, therefore
reducing the needs for animal studies. Reducing in-vitro artefacts would
enable a more reliable prescreening of potential cartilage repair
therapies.
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60 Publisher, aofoundation.org, Davos, Switzerland
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3 We thank the reviewers for their helpful comments. Based on the reviews we have substantially
4 shortened the text and polished the language, reducing from 20 to 13 pages. So much so, in addition
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to the tracked changes, we have also uploaded a clean version as supplementary data. Each
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comment is answered in detail below.
8 We hope the changes receive positive feedback.
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10 Reviewer: 1
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12 Introduction: Abstract
13 Line 11: Please clarify if you mean shear and dynamic compression are forces or motions.
14 Introduction
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17 We have corrected this to "forces".
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19 Page 3, Line 45: Please check reference formatting. All other references seem to be formatted
20 differently.
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22 We have corrected the formatting.
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Page 4, Line 10: Please correct this sentence. I believe a word is missing after ‘Indeed’.
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27 We have added the missing word "it"
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29 Discussion - Errors/comments: Page 14 Line 32: It should be ‘hBMMSCs’ instead of ‘hBMMCs’.
30 We have made the correction
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32 Page 17, Line 34: Please clarify this sentence. I believe a word is missing after ‘study.’
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This has been corrected and the whole paragraph shortened in line with the request from
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Reviewer 2.
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37 Page 17, Line 43: Please include correct reference.
38 We have removed this aspect as part of the discussion shortening.
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40 Page 18, Line 60: I believe there is a word missing at the end of the sentence.
41 We have added this missing "pericellular matrix".
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Conclusion - Errors/comments: Appropriate.
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46 References - Errors/comments: Please make sure references throughout the manuscript are
47 consistent with the journal's format.
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49 We have checked and made the required changes throughout the document.
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51 Illustrations- Quality: Appropriate.
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General Errors/comments not listed in the sections above:
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56 MSCs were isolated from older donors. Do you expect that results would be different from younger
57 donors? I observed that in Figure 8 Donor 1 had no gag staining in the HA+Load compared to the
58 other donors. I understand that this was not the focus of the manuscript, however, it would be
59 helpful to discuss within the Discussion.
60 The reasons for donor variation is still unclear but is a very interesting aspect. To the discussion we
have added " The matrix production and deposition was donor dependent, with donor 1 in
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3 particular being weak in histological staining. This could be related to the donor age, this
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study used older donors, or the variation may be a factor of the monolayer expansion/
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selection. "
7 Please justify how the mechanical loading regimen was selected including loads and duration.
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9 Similar to reviewer 2, we have added to the discussion that the 1 hours loading regime is a
10 limitation of the bioreactor. We can load 4 samples per hour, then need to exchange samples in
11 the bioreactor. Including the time needed to exchange samples, loading 16 samples for 1 hours a
12 day requires 5 hours.
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If the paper is acceptable (1 or 2 - See Recommendations below), please assess the
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16 significance/character of this paper.: Original research report of general interest
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18 In addition to reviewing the paper as critically as you would for any other respected journal, we
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19 desire relevant questions such as may arise at a conference where this paper is presented as written.
20 Questions which bring out additional information or which challenge the authors' approach, findings,
21 or conclusions, are particularly welcome. While some of these questions may be attended to by
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22 appropriate text changes, MOST questions and authors' replies will be published with the paper as
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"Discussion with Reviewers" (with the reviewers named, as thanks for reviewing, unless requested
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specifically not to be named.
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27 Please add your questions here. (Please insert N/A into the text box below if you do not answer 1
28 or 2):
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30 The literature suggests that in vitro studies that apply multi-axial mechanical loads to MSCs promotes
31 chondrogenesis. However, rehabilitation protocols that include early weight bearing and aggressive
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32 strengthening following a procedure such as microfracture are more likely to produce fibrocartilage.
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Please comment on how we can better translate these important in vitro findings to better
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understand the suboptimal in vivo outcomes for patients.
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37 This is a clear paradox in the field. Currently we do not have a definite answer. However, our
38 working hypothesis is based on the localized activation of TGFb protein. We have shown this under
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39 complex load and this offers a potential coupling of local mechanical strain to a local biological
40 response. It would also explain why the concentration of TGF in the mechanically stimulated
41 samples is so low and yet a chondrogenic response is observed. We also have data showing that
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the material properties need to reach a threshold of stiffness, below which this activation cannot
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44 occur. Our hypothesis with microfracture is that the initial marrow clot is too soft to allow
45 localized mechanically induced TGFb activation. It could also offer some insight into why
46 microfracture becomes less effective the larger the defect, as the soft surface area increase, the
47 localized TGFb activation decreases. We believe adding a stiffer macroporous support, a role
48 played by the polyurethane in this study, would increase TGF activation and potentially improve
49 outcomes.
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Reviewers are named in the published paper by default. Please confirm you agree, or request
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anonymity.: I request my review remains anonymous
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56 Reviewer: 2
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58 Recommendation: 2. Major textual revision required for acceptance.
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60 Comments:
(There are no comments.)
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4 Additional Questions:
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Original findings: YES
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8 Aim: OK.
9 The authors have previously shown that a combination of shear and compressive loading enhances
10 chondrogenesis by stimulating the production and activation of TGF-beta. They now wish to see
11 whether the addition of HA further improves chondrogenesis.
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13 Introduction: OK
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Materials & Methods
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17 Described well enough to allow repetition of the work: YES
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19 Materials and methods comments:
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21 Results - Errors/comments: 1. Were the authors surprised that their mechanical loading regimen,
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22 which included shear, did not induce PRG4? Shear is well known to increase expression of this
23 molecule.
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26 Author response: Shear is known to increase PRG4 and we have shown this in previously published
27 work from our group 1. Within this study there was also a tendency to an increase, in particular at
28 day 7.
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29 Donor 1 Donor 2 Donor 3
30 DMEM static 4.883731 170.3183 59.39319
31 DMEM load 847.2293 319.7205 329.9543
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32 HA+ static 5.490989 174.8205 27.65223
33 HA+ load 444.6344 141.4597 307.0326
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35 Due to the variation the changes did not reach significance. In addition to the usual donor
36 variation, one aspect that leads to further variation, is the cell distribution within the scaffold. If
37 one sample has a lower proportion of the cells near the surface, less experience the shear and the
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PRG4 upregulation is not as great. We have started to asymmetrically seed scaffolds as it also
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40 dramatically improves the chondrogenic response 2, but unfortunately, we did not adopt that
41 approach in this study.
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44 2. Figures 5 and 6 are not cited in the results section text.
45 The Citation has been added.
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3. Do figures 5 and 6 show the same data? If so, please pick one. (I prefer figure 6).
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49 Figures 5 and six do show the same data, albeit that figure 5 enables the significant differences to
50 be more easily highlighted. As it is more informative, we have kept figure 5 and added the final
51 summary aspect from figure 6 for illustrative purposes.
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53 4. It appears from figure 3 that a greater percentage of the total GAG is soluble in the absence of HA.
54 Is it possible that newly formed aggrecan is being broken down in the absence of HA? This would
55 explain why there is a difference in total GAG between the two groups, even though aggrecan mRNA
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levels are not statistically different (figure 2). HA may inhibit the induction or activities of e.g.
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ADAMTS, MMP-3 etc.
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60 The frequent lack of correlation between aggrecan mRNA expression and produced GAG is
perplexing. We have looked at various factors over the years and have not yet found a conclusive
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3 answer. Based on the suggestion of the reviewer, we have looked into this in more detail. We
4 assessed mRNA expression of MMP3, ADAMTS4 and ADAMTS 5. For both MMP3 and ADAMTS4 we
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detected significant differences between loaded and unloaded samples, with load inducing an
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increase. The addition of HA had no effect. No difference in ADAMTS5 expression was observed.
8 The data has been included as new figure 3. We realize the format of the graph is different to the
9 other figures, but unfortunately the PhD student responsible for the project has been in intensive
10 care since November. The PI has been making all the changes and has been unable to format the
11 graph in the same way.
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13 5. Figure 7 gives a beautiful confirmation of the ability of mechanical loading to induce and activate
14 TGF-beta. I am struck by how little TGF activation is needed (5% or less) to enhance chondrogenesis
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(the y-axis of panel 7D is mis-labelled with an additional (pg/ml)). It is also striking how much more
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17 potent the endogenously synthesized TGF-beta is compared to recombinant TGF-beta. The former
18 seems to be active at a few hundred pg/ml whereas the standard chondrogenesis assay usually uses
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19 around 10 ng/ml TGF-beta.
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21 Thank you for the comment. This has been shown in multiple studies and it is an exciting aspect of
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22 the model. It is possible that the local production/ activation enhances the potency due to
23 proximity of the active molecule to the cell, but this is a difficult aspect to investigate. In addition,
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TGF is known to be a sticky protein, a proportion of the added protein is likely to be lost by binding
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26 plastic etc.
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28 6. The authors used a loading regimen of 1 hour a day, 5 days a week. Do they think that a different
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29 regimen might enhance chondrogenesis even more (figure 8)?
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31 Yes. This is a limitation of the current bioreactor, which can only load 4 samples at a time. We are
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32 in the final stages of developing a 16 well bioreactor to enable more flexibility in the loading
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protocols. We have added the following to the discussion " A limitation of the current study is
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the application of load for 1 hour a day. In part, this is due to limitations within the bioreactor
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36 system itself. A high throughput bioreactor system is currently under development to enable
37 the study of loading duration on the differentiation process."
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39 Discussion - Errors/comments: The discussion is far too long - it occupies about 50% of the entire
40 text. It reads as if it has been cut-and-paste from a PhD thesis.
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Please reduce to 5 pages or less; if possible, please do not repeat the results but place them in the
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44 context of how they advance the field.
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46 The reviewer has a keen eye. The discussion was reworked from a PhD thesis chapter. We have
47 made substantial changes and reduced the text as requested.
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1 Li, Z., Kupcsik, L., Yao, S. J., Alini, M. & Stoddart, M. J. Mechanical Load Modulates
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Chondrogenesis of Human Mesenchymal Stem Cells through the TGF-beta Pathway.
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55 J Cell Mol Med 14, 1338-1346 (2010).
56 2 Gardner, O. F. W. et al. Asymmetrical seeding of MSCs into fibrin-poly(ester-
57 urethane) scaffolds and its effect on mechanically induced chondrogenesis. Journal
58 of tissue engineering and regenerative medicine 11, 2912-2921,
59 doi:10.1002/term.2194 (2017).
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4 Sodium Hyaluronate supplemented culture media combined with
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6 joint-simulating mechanical loading improves chondrogenic
7 differentiation of human Mesenchymal Stem cells
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11 Graziana Monaco 1,2, Alicia Jennifer El Haj3, Mauro Alini1 and Martin James
12 Stoddart1,2*
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14 1AO Research Institute Davos, Davos, Switzerland, 2 Guy Hilton Research Centre, School of
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Pharmacy and Bioengineering, Keele University, Stoke-on-Trent, Staffordshire ST4 7QB,
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17 United Kingdom, 3Healthcare Technology Institute, Institute of Translational Medicine,
18 University of Birmingham, B15 2TT, United Kingdom
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20 * Correspondence:
21 Martin James Stoddart
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22 martin.stoddart@aofoundation.org
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24 Keywords: Hyaluronic acid, Mesenchymal Stem cells, chondrogenic differentiation, articular
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cartilage, hypertrophy, joint simulating bioreactor, mechanical loading, in vitro model, culture
27 media, TGF-β1
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3 Abstract
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In-vitro models aim to recapitulate the in vivo situation. To more closely mimic the knee joint
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7 environment, current in vitro models need improvements to reflect the complexity of the native
8 tissue. High molecular weight hyaluronan (hMwHA) is one of the most abundant bioactive
9 macromolecules of in healthy synovial fluid, while shear and dynamic compression are two
10
11 joint-relevant mechanical motionsforces.
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13 The present study aimed to investigate the concomitant effect of joint-simulating mechanical
14 loading (JSML) and hMwHA supplemented-culture media on the chondrogenic differentiation
15 of primary human bone marrow-derived mesenchymal stem cell (hBMMSC).
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17 hBMSC chondrogenesis was investigated over 28 days at the gene expression level and total
18
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19 DNA, sulphated glycosaminoglycan, TGF-β1 production, and Safranin O staining were
20 evaluated.
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22 The concomitant effect of hMwHA culture media and JSML, significantly increased cartilage-
23 like matrix deposition and sulphated glycosaminoglycan synthesis, especially in during early
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chondrogenesis. A stabilization of the hBMSC-derived chondrocyte phenotype was observed
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26 through the reduced upregulation of the hypertrophic marker collagen X and an increase in the
27 chondrogenic collagen type II/X ratio.
28
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30 The concomitant effect of A combination of JSML and hMwt HA media, better reflects the
31 complexity of the in vivo synovial joint environment. Thus, JSML and hMwt HA media will be
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32 two important joint-related features to be simultaneously introduced for within the joint-related
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culture models to more accurately predict the in vivo outcome, thus therefore reducing the needs
for animal studies. Reducing in-vitro artefacts would enable a more reliable prescreening of
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36 potential cartilage repair therapies.
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3 Introduction
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Trauma, osteoarthritis and osteochondritis are the most common causes of cartilage damage,
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7 leading to pain, swelling and impaired movement of the joint (Madry et al., 2011). The demand
8 for effective treatment strategies to treat cartilage lesions is continually increasing. However,
9 current therapies have considerable limitations, prompting the development of novel cartilage
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11 tissue engineering approaches.
12 Autologous Chondrocytes Implantation (ACI) and Matrix-assisted Autologous Chondrocytes
13 Implantation (MACI) have been approved by the Food and Drug Administration (FDA) and by
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15 the European Medicines Agency (EMA) as Advanced-Therapy Medicinal Products (ATMPs)
16 (Brittberg, 2010; Brittberg et al., 1994; Makris et al., 2015). However, for both treatments,
17 donor site morbidity due to the required cartilage biopsy remains an issue, as does the small
18
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19 size of harvestable cartilage that is associated with a low chondrocyte yield and limited in-vitro
20 expansion potential (Brittberg, 2010; Brittberg et al., 1994; Erggelet et al., 2003; Knecht et al.,
21 2007). In addition, the need to expand chondrocytes in monolayer increases the risk of
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23 dedifferentiation into fibroblastic cells (Benya and Shaffer, 1982; Hegewald et al., 2004).
24 Due to the limited supply of autologous chondrocytes for transplantation procedures, much
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25 attention was drawnhas focused on to mesenchymal stem cells (MSCs), which is also the cell
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27 type involved in the regeneration processes when microfracture, a leading surgical technique
28 for healing chondral defect, is used (Kang et al., 2008; Oussedik et al., 2015; Steadman et al.,
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29 2001).
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Particularly, human bone marrow-derived mesenchymal stem cells (hBMMSCs) represent an
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32 attractive alternative cell source to autologous chondrocytes since they can be easily isolated
33 from bone marrow aspirates with limited donor site morbidity and following expansion they
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continue to maintain multilineage potential (Gardner et al., 2013; Hegewald et al., 2004).
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36 Indeed, hBMMSCs are well investigated in the clinical setting, are the best characterized and
37 can be used for subchondral bone and overlying articular cartilage repair (de Vries-van Melle
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et al., 2014; Nejadnik et al., 2010; Parekkadan and Milwid, 2010; Wakitani et al., 1994;
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40 Wakitani et al., 2002).
41 However, one of the main challenges of in vitro hBMMSC chondrogenic differentiation is the
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lack of a suitable culture environment that would better reproduce the in vivo physiological
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44 conditions and, in so doing, may prevent or reduce the progression of MSC-derived
45 chondrocytes through hypertrophic differentiation that causes the neo-formed cartilage to
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undergo endochondral ossification (Johnstone et al., 1998a).
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48 Due to the discrepancies that exist, current in vitro models need to be improved to reflect the
49 complexity of the joint environment found in vivo, thus aiming to reduce the gap between in
50 vitro and in vivo results. More accurate in vitro models will be crucial to prevent in vitro
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52 artefacts and to produce more reliable results, enabling more accurate in vitro prescreening of
53 potential cartilage repair therapies, thus reducing the number of animals used for in vivo studies.
54 To improve the current in vitro models, characteristic features that would help mimic the native
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56 tissue, need to be introduced into the culture system. For this purpose, it is necessary to consider
57 that articular cartilage or hyaline cartilage due to its unique molecular composition and
58 structure, plays an essential role in joint lubrication and impact absorption lining the articulating
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60 surface of bones (Hosseini et al., 2014; Responte et al., 2007; Sophia Fox et al., 2009; Wu and
Ferguson, 2017). During joint articulation, mechanical cues profoundly affect cell and tissue
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3 responses influencing the homeostasis of healthy tissue or leading to degeneration of articular
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5 cartilage (Bader et al., 2011; Grodzinsky et al., 2000; Sophia Fox et al., 2009).
6 Therefore, several studies aim to clarify the mechanisms involved in cell response under loading
7 conditions. hBMMSCs have been utilized in multiple mechanical loading studies (Schatti et al.,
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2011; Schumann et al., 2006).
10 Indeed, it has been previously observed that complex multiaxial load in vitro, by mimicking the
11 mechanical motion of an articulating joint, induced gene expression and protein production of
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endogenous TGF-β1 and TGF-β3, key molecules that address the chondrogenic differentiation
14 of hBMMSCs (Li et al., 2010b). In addition, mechanical loading, not only induced an increased
15 TGF-β expression but also directly led to the activation of latent endogenous TGF-β (Li et al.,
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2010). Mechanical loading therefore enhances MSC chondrogenic differentiation by
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18 resembling the in vivo conditions and inducing a more physiological production and activation
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19 of TGF-β by hBMMSCs.
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High molecular weight hyaluronic acid (105-107 Da) (hMwt HA), one of the major components
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22 of healthy synovial fluid and cartilage extracellular matrix, plays an important role when
23 mechanical loading is applied, mainly by protecting the opposing articular cartilage surfaces,
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improving joint lubrication and acting as shock absorber (Hegewald et al., 2004).
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26 On the other hand, hyaluronic acid which resides in the synovial fluid, is also involved in the
27 nutrient and waste transport towards and from cartilage tissue and is able to maintain water
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homeostasis inside the joint due to its excellent osmotic buffering property (Laurent et al., 1996;
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30 Lynch et al., 1998; McDonald and Levick, 1995).
31 Hyaluronic acid, as well as synovial fluid, were shown in vitro to have the potential tocan induce
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32 in vitro chondrogenic differentiation in chicken limb bud bioassays (Kujawa et al., 1986;
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34 Maleski and Knudson, 1996; Rodrigo et al., 1995). In addition, in vivo during microfracture
treatment, mesenchymal stem cells (MSCs) that reside in the bone marrow cavity of the
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36 subchondral bone, are exposed to HA contained within synovial fluid (Kang et al., 2008;
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38 Oussedik et al., 2015; Steadman et al., 2001).
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39 Therefore, hyaluronic acidHA has been extensively used as target molecule for scaffold or
40 hydrogel manufacturing, alone or in combination with MSCs to reproduce engineered cartilage
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with native structure or to enhance the repair of osteochondral defect (Gallo et al., 2019;
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43 Huerta-Ángeles et al., 2018; Li et al., 2018; Radice et al., 2000; Solchaga et al., 2000).
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45 However, few studies describe the use of hyaluronic acidof HA as an exogenous media
46 supplement to promote the chondrogenic differentiation of MSCs (Hegewald et al., 2004;
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Monaco et al., 2020). In a previous study the effect of high molecular weight hyaluronic acid
49 was investigated as a media supplement on the chondrogenic differentiation of hBMMSCs from
50 patients (Monaco et al., 2020). However, this study investigated only the biological activity of
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hyaluronic as media supplement under static conditions.
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Therefore, the present study hypothesizes a potential beneficial synergistic effect on
55 chondrogenic differentiation of hBMMSCs during joint-simulating mechanical load in the
56 presence of high molecular weight hyaluronic acid supplemented culture media.
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Therefore, tThe purpose of the present study was to determine if a physiological concentration
59 (2 mg/ml) of exogenous high molecular weight HA 1.8MDa (hMwHA) administered as media
60 supplement in chondropermissive culture media, combined with joint-simulating mechanical
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3 loading, would allow a more physiologically-driven chondrogenic differentiation of MSC
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5 seeded within Polyurethane:fibrin-based construct (Figure 1).
6 By including these factors into our in vitro culture model, we would create a culture system that
7 better reflects the complexity of the in vivo joint environment. We expect that the newly
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developed culture system, by better approximating in vivo, would also offer more reliable
10 results for the screening of potential cartilage repair therapies.
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3 1. Materials and Methods
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2.1 Poly(ester-urethane) scaffolds preparation
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7 Poly(ester-urethane) porous sponges (PU) were prepared using hexamethylene diisocyanate,
8 poly (1-caprolactone) diol and isosorbide diol (1,4: 3,6-dianhydro-D-sorbitol) via a salt leaching-
9 phase inverse technique (Gorna and Gogolewski, 2002). With this procedure interconnected
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macroporosity ranging from 90 to 300µm has been uniformly achieved within the sponge. The
12 PU sponge was cut by water-jet (CUTEC AG, Basel, Switzerland) producing cylindrical
13 scaffolds (8mm dimeter x 4 mm height), sterilized in a cold cycle at 37°C via ethylene oxide and
14 degassed under vacuum for six days before use.
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16 Isolation of human bone marrow derived MSCs
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18 Bone marrow was obtained with full ethical approval (KEK-ZH-NR: 2010–0444/0) and the
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19 written consent from patients undergoing routine operations (Table 1).
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21 The MSCs were isolated from three different marrow aspirates (two male 1967 50 and 65 year
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22 oldand 1951, one female 195462 years old) using Ficoll density separation (Sigma-Aldrich,
23 Buchs, Switzerland).
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Mononuclear cells were collected from the interphase and the adherent cell fraction was seeded
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26 at a density of 50,000 cells/cm2 and left to attach for 96 hrs in alpha minimum essential medium
27 (αMEM) (Gibco, Carlsbad, CA, USA), 10% MSC tested fetal bovine serum (FBS) (Pan Biotech,
28
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Aidenbach, Germany), 5 ng/ml basic fibroblast growth factor (bFGF) (Peprotech, Rocky Hill,
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CN, USA) and 1% penicillin/streptomycin (Gibco). When the majority of colonies were
31 confluent, the cells were passaged and seeded into fresh flasks at a cell density of 3,000 cells/cm2.
The chondrogenic potential of each donor was confirmed using standard techniques.
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The hBMMSCs isolated from each donor were used separately in three independent experiments.
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36 Scaffold seeding and chondrogenic differentiation
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38 hBMMSCs at passage 3 were trypsinized at 80% confluence, suspended in a 150 μl fibrinogen-
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39 thrombin-solution and evenly seeded at a cell density of 5x106 cells/150 μl in cylindrical (8mm
40 x 4mm) macroporous polyurethane (PU) scaffolds. The constructs were fed with two different
41 media for 28 days. Control medium was serum free basal medium containing DMEM high
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42 glucose, supplemented with 1% ITS+, 1% Pen/Strep, 1% non-essential amino acid, 50 μg/ml
43 ascorbate-2-phosphate, 5 μM ε-amino-caproic acid (EACA), 10-7M dexamethasone (DMEM).
44
45
This media was further supplemented with 0.2% 1.8 MDa HA (HA+) (Stanford Chemicals) to
46 simulate the synovial fluid concentration under normal conditions (2.3 mg/ml) (Fam et al., 2007).
47 The culture medium was refreshed every second day, and conditioned medium was collected for
48 analysis.
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52 Mechanical Loading
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54 Control constructs for both culture media (DMEM and HA+ groups) were kept under static
55 culture for the entire culture period (4 weeks). A custom-made joint-simulating bioreactor based
56 on tribological principals was used to exert the multi-axial loading on the surface of the
57 experimental constructs (Wimmer et al., 2004). Loaded constructs were exposed to 20 cycles
58 of 10% compression superimposed on top of a 10% pre-strain and shear loading (± 25°) at 1Hz
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for 1 h a day, five times a week. The application of multi-axial mechanical load to fibrin–
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3 poly(ester-urethane) constructs in this system has been described previously by Zahedmanesh
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et al.(Zahedmanesh et al., 2014).
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7
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9 Gene expression analysis: RNA isolation, cDNA synthesis, Real Time qPCR
10 After 0, 7, 14, 21 and 28 days of chondrogenic culture, constructs were harvested, and total RNA
11 was isolated using TRI Reagent (MRC, Cincinnati, OH/ Molecular Research Centre Inc).
12 TaqMan reverse transcription was then performed using 1 µg of total RNA sample, random
13 hexamer primers and TaqMan reverse transcription reagents (Applied Biosystems, Carlsbad, CA,
14
USA).
15
16 Real-time PCR was performed using the QuantStudio 6 Flex real-time PCR system (Applied
17 Biosystems). A panel of human genes associated with chondrogenic markers (COL2A1, ACAN,
18 Sox9), the hypertrophic marker COL10A1, osteogenic markers (RunX2, ALP), Hyaluronan
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19 receptors (CD44, RHAMM), and mechanically responsive genes (PRG4, COMP) were
20 investigated.
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22 Primers for RPLP0, COL2A1, COL10A1, ACAN and RunX2 mRNA were synthesized by
23 Microsynth AG (Balgach, Switzerland) (Table 2). Primers for Sox9, ALP, CD44, RHAMM,
24 PRG4, and COMP, MMP3, ADATS4 and ADAMTS5 were purchased from Applied
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25 Biosystems (Warrington, United Kingdom) (Table 3)
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27 Relative quantification of target mRNA was determined according to the comparative CT method
28 with hRPLP0 as endogenous control. In addition, the level of gene expression for each gene was
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29 determined relative to day 0 monolayer via a ΔΔCT comparison (Table 2 and 3).
30
31
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Sulphated glycosaminoglycan and DNA quantification
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34 After 28 days of culture, constructs were digested with 1 ml proteinase K (0.5 mg/ml) at 56°C
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35 for 16 hours. Total DNA content was measured spectrofluorometrically following reaction with
36
Bisbenzimide Hoechst 33258 dye (Polysciences Inc., Warrington, PA, USA) with purified calf
37
38
thymus DNA as standard (Lubio Science, Luzern, Switzerland) (Labarca and Paigen, 1980).
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39 Sulphated glycosaminoglycans (GAG) retained within the scaffolds was determined by a direct
40 spectrophotometric microassay according to the dimethylmethylene blue dye method (Sigma-
41
Aldrich, Buchs, Switzerland) at pH 1.5, using bovine chondroitin 4-sulfate sodium salt from
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43 bovine trachea (Fluka, St Louis, MO, USA) (Farndale et al., 1986). Total GAG content of the
44 culture media was also measured to assess the release of matrix molecules from the constructs.
45 All samples containing hyaluronan were blanked with media containing 0.2% hyaluronan and
46 DMMB at pH 1.5 was used to eliminate background due to the residual interaction between
47 DMMB and Hyaluronan.
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49
ELISA TGF-β1 quantification
50
51 Both the amount of total TGF-β1 and active TGF-β1, in collected culture media was quantified
52 using the human TGF-β1 DuoSet ELISA (R&D systems, Minneapolis, USA). In order to
53 measure the total amount of TGF-β1 in each sample an acidic activation step was performed as
54
55
per the manufacturer's instructions. Analyses of the samples without this activation step provided
56 the amount of active TGF-β1 within the sample.
57
58 Histology and Staining
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3 After 28 days of culture, the constructs were fixed in 70% methanol and 10 µm specimen sections
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were cut by cryostat, stained with Safranin O and counterstained with Fast Green to detect
5
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proteoglycan presence and proteoglycan-depleted, collagen-rich areas.
7
8
9
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11 Statistical analysis
12
13 The data were produced from three individual experiments, each carried out with hMSC from a
14 different donor. All experiments were performed in triplicate and quadruplicate for each group
15 at different timepoints in order to reduce methodological variability. Each measurement was
16 performed in duplicate. Analyses were done between the appropriate control group and treatment
17 groups as well as between different treatment groups, using one way or two ways ANOVA with
18
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Tukey’s or Sidak's Posthoc testing as required. A significance level of p < 0.05 was applied and
19
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data are presented as Mean and SD. Analyses were carried out using the GraphPadPrism 8.1.0
21 software (GraphPad Software Inc., La Jolla, CA, USA).
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3 2. Results
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Gene expression analysis
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7 A panel of genes associated with chondrogenic differentiation (Collagen type II, Aggrecan,
8
9
Sox9) were investigated, as well as hypertrophy associated Collagen type X associated with
10 hMSC hypertrophic differentiation, genes associated with osteogenic differentiation (Runx2,
11 ALP) and mechanical loading (PRG4, COMP) (Figure 2). To gain further understanding of the
12 underlying mechanism, the hyaluronan receptors (CD44, RHAMM) and genes associated with
13 matrix breakdown (MMP3, ADAMTS4 and ADAMTS5) were also investigated. All of the
14 donors investigated displayed similar trends in the levels of gene expression with a varying
15
degree of magnitude. With the chondrogenic markers, an overall upregulation of the Collagen
16
17 II, Aggrecan and Sox9, involved in chondrogenic differentiation was observed under loading
18 conditions. The mechanical loading also induced an increased expression of Collagen 10.
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19
20 In this study, cConcomitant loading and hyaluronic acidHA supplemented media lead to a
21 significant Collagen II upregulation at day 14 and 21 compared with DMEM (*pPage 15 of 71 EUR CELL Mater: True Open Access ecmjournal.org
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3 observed for the DMEM loaded group. In addition, the average value of PRG4 expression in
4
the HAHA+ loaded group was never significantly different from the static groups. A significant
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PRG4 upregulation was observed at day 7 in the DMEM loaded compared with the DMEM
7 static (*pEUR CELL Mater: True Open Access ecmjournal.org Page 16 of 71
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3 GAG release observed for thewith HAHA+ media was further enhanced when mechanical
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loading was applied. Indeed, the HAHA+ loaded group at day 3 showed significantly higher
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GAG per media not only compared with DMEM static (****pPage 17 of 71 EUR CELL Mater: True Open Access ecmjournal.org
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3 GAG production and deposition. Although the magnitude of sGAG production was donor-
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dependent, the trend observed in the present study was reproducible and robust.
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ELISA TGF-β1
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Total TGF-β1, active TGF-β1, active cumulative TGF-β1 1and percentage of active TGF-β1
10 were analysed to understand ifassess whether HAHA+ media under mechanical loading
11 affected TGF-β1 expression and activation during hBMMSC chondrogenic differentiation
12 under static or loaded conditions..
13
14 Under static conditions, a significantly higher TGF-β1 level production was observed for both
15 culture media at day 7 when compared with all the other timepoints investigated (day 14, 21,
16
28) within the same static groups (DMEM or HA media) (Figure 7A). Similarly, loaded samples
17
18 had a peak of TGF-β1 production on day 7. This suggests that within the first week of MSC 3D
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19 culture, TGF-β1 was actively produced by MSCs in both static and loaded conditions, with a
20 greater production observed under loading. Then, aA lack of mechanical load in the static
21 conditions led to a significant decrease in TGF-β1 production which decreases over time (****p
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3 Taken together, the TGF-β1 results suggest that under static conditions significantly less TGF-
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β1 was produced than under loaded conditions, independent of the media and timepoint
5
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investigated. However, mechanical loading promoted both the production and the activation of
7 TGF-β1, independently of the media and timepoint investigated.
8
9 Histology & Safranin O/Fast green staining
10
11 In order to show the deposition of sGAG, differentiated constructs were stained with Safranin
12 O and counterstained with Fast green after 28 days of chondrogenesis (Figure 8A-L). DMEM
13 culture media under static conditions showed no ECM deposition with any donor. Addition of
14 HA under static conditions improved ECM deposition in donor 2 (Figure 8H) and partly also
15
in donor 3 (Figure 8I). Mechanical loading alone increased ECM deposition and safranin O
16
17 staining along the upper surface of the PU:fibrin scaffolds fed with DMEM only in donor 2.
18 The concomitant effect of HAHA+ culture media and mechanical loading notably increased the
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19 ECM deposition and Safranin O staining along the upper surface, in two donors out of 3 (donor
20 2 and 3). In all conditions, donor 1 was not affected by any of the treatments investigated.
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3 Discussion
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5
The simultaneous investigation of bioengineered culture media composition and loading
6
7 motion, in the absence of exogenous growth factors, can provide more physiological conditions
8 to improve our understanding of human bone marrow-derived MSCs chondrogenesis and
9 functional cartilage-like tissue development.
10
11 Within joints in vivo, and particularly within the knee, the forces that most cartilage is exposed
12 to during day to day physiological movements are a combination of compression, caused by
13
14
gravitational or muscular loading, and shear stress generated by the movement of the two
15 articular surfaces across each other and the movement of synovial fluid across the surface of
16 the tissue.
17
18 Therefore, mechanical loading and particularly multiaxial shear and compression are two
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19 important cues addressing MSC chondrogenesisfate and have been shown. Interestingly, when
20 these two loading motions are applied to MSCs seeded within fibrin:polyurethane scaffolds, the
21
cells undergo to induce chondrogenesis in the absence of any exogenous growth factors e.g.
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23 TGF-β1 (Kupcsik et al., 2010; Li et al., 2010; Li et al., 2010b; Neumann et al., 2013). The up-
24 regulation of gene markers associated with MSC chondrogenic differentiation such as collagen
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25 type II and aggrecan, as well as the deposition of collagen type II cartilage-like matrix is also
26
27 been enhanced under mechanical loading (Li et al., 2010a).
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On the other hand, the formulation of a suitable culture media to develop in vitro tissue-tailored
30 constructs has been attempted in several ways (Hegewald et al., 2004; Monaco et al., 2020) .
31 Previous findings demonstrated that the exogenous supplementatisupplementingon culture media
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with of exogenous hyaluronic acidHA within the culture media enhanced the chondrogenic
33
34 differentiation of equine MSCs in pellet culture and human bone marrow MSCs in 3D tissue
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35 engineered constructs, through an improvement of thewith increased extracellular matrixECM
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production and reduction reduced of the MSC hypertrophic differentiation y (Hegewald et al.,
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38 2004; Monaco et al., 2020). An increased expression of Sox9 and Aggrecan has been
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39 demonstrated when supplementing culture media with TGF-β3 and using HA as a culture plate
40
coating (Bhang et al., 2011). The improved chondrogenic differentiation by combining TGF-β3
41
and a bioactive molecule such as HA was shown to be additive (Bhang et al., 2011). Previous
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43 studies also demonstrated that hMwt HA administered at a physiological concentration (2mg/ml)
44 as a media supplement in chondropermissive culture media, exerted a beneficial effect on the
45
46 expression of Sox 9 and aggrecan but did not induce a significant increase of collagen type II
47 gene expression under static conditions (Monaco et al., 2020). Previous studies carried out
48 underFurthermore, addition of static conditions and investigating the use of HA asto the media
49
50 supplement or as part of tissue engineered constructs describedled to a similar reductioned of
51 collagen type X expression that was also dose dependent (Amann et al., 2017; Hegewald et al.,
52 2004; Monaco et al., 2020).
53
54
55
56
57 However, previous studies focused on the investigation of one parameter, evaluating the impact
58 of mechanical loading alone or the effect of the culture media composition through hyaluronan
59
60 supplementation (Hegewald et al., 2004; Monaco et al., 2020).
14
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3
4
5 The present study aims aimed to investigate the synergistic effects of exogenous high molecular
6
7 weight hyaluronic acid and joint-simulating mechanical loading on the chondrogenic
8 differentiation of human bone marrow-derived MSCs over 28 days.
9 The physiological concentration of 2mg/ml and the high molecular weight HA (of 1.8MDa),
10
11
were selected since they have been shown to enhance chondrocyte function under static and
12 mechanical loading condition respectively for hBMMSCs and for cartilage tissue development
13 in a model based on chondrocytes isolated from fetlock joints (Monaco et al., 2020; Wu et al.,
14
15
2017).
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18
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The inclusion within the same culture system of hyaluronic acid media supplementation and
19
20 mechanical loading established within the present culture model represents a more physiological
21 environment that might improve the reliability of in vitro hBMMSC chondrogenesis carried out
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within a 3D tissue engineered construct, therefore reducing the in vitro artefacts and thus the gap
23
24 between in vitro and in vivo.
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25 hBMMSC chondrogenic differentiation was carried out over 28 days and the development of
26
engineered cartilage constructs was investigated at the gene and protein level at different
27
28 timepoints both under static or loading conditions, with or without hMwt HA supplemented
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29 chondropermissive culture media.
30 The present study demonstrated that treating hBMMSCs, isolated by ficoll-paque density
31
gradient, monolayer expanded until passage 3 and seeded within a 3D macroporous
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33 polyurethane:fibrin, with hMwHA under joint-simulating mechanical loading significantly
34 improved chondrogenic differentiation at the gene expression, protein deposition and histological
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36 levels.
37 Particularly, when combining hMwHA with mechanical loading, we observed: 1) at the gene
38 expression level, a reduced expression of the hypertrophic marker collagen type X and lubricin,
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40 as well as an upregulation of the chondrogenic markers collagen type II, aggrecan and collagen
41 type II/collagen type X ratio; 2) a more marked extracellular matrix deposition detected both
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42 through sGAG assay and histological staining; 3) a significantly higher sGAG production and
43
44 release into the culture media particularly marked within the first week of MSC chondrogenesis;
45 4) Slightly but not significantly higher average levels of total TGF-β1; 5) consistent and
46 reproducible trends for three different human bone marrow MSC donors over 28 days of
47
48
chondrogenic culture.
49
50 hMwHA media under joint-simulating mechanical loading generates a stable chondrocyte
51
52
phenotype by reducing the hypertrophic marker collagen type X gene expression and increasing
53 the collagen type II/X ratio. Several studies have demonstrated the beneficial effect of joint
54 simulating mechanical loading on the chondrogenic differentiation of hBMMSCs. Particularly,
55
56
previous studies proved that mechanical loading alone was sufficient to increase gene expression
57 of the chondrogenic markers collagen II, aggrecan and Sox9 (Li et al., 2010a; Li et al., 2010b).
58 Aggrecan gene expression has been previously described as mechanosensitive (Grad et al.,
59
2006a; Grad et al., 2006b).
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3 On the other hand, hyaluronic acid has been previously used in several hMSC chondrogenic
4
5 differentiation studies conducted under static conditions (Avenoso et al.; Gallo et al., 2019;
6 Hegewald et al., 2004; Monaco et al., 2020). One of these studies showed an increased expression
7 of Sox9 and Aggrecan when supplementing the culture media with TGF-β3 and using hyaluronic
8
9
acid as coating for the culture plates (Bhang et al., 2011). The improved chondrogenic
10 differentiation by introducing a soluble growth factor such as TGF-β3 and a bioactive molecule
11 such as hyaluronic acid was shown to be additive (Bhang et al., 2011). Previous studies also
12
13
demonstrated that hMwt HA administered at a physiological concentration (2mg/ml) as a media
14 supplement in chondropermissive culture media, exerted a beneficial effect on the expression of
15 Sox 9 and aggrecan but did not induce a significant increase of collagen type II gene expression
16
under static conditions (Monaco et al., 2020).
17
18 The present work confirmed previous findings on the positive effect of joint-simulating
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19 mechanical loading in enhancement of collagen type II, aggrecan and Sox9 gene expression. In
20
addition, in the present study, the concomitant effect of hMwt HA supplemented media and joint-
21
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22 simulating mechanical loading, investigated at the same time, lead to a further increase of
23 collagen type II and aggrecan gene expression. Particularly at day 14 a more marked collagen
24
type II gene expression and a significant increase in aggrecan gene expression was observed
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26 when hMwt HA and mechanical loading were applied. The addition of hMwt HA media to the
27 loading environment induced an earlier upregulation of collagen type II and aggrecan gene
28
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expression. This earlier upregulation of chondrogenic markers was consistently observed in all
29
30 three different donors and would be beneficial for cartilage-like matrix production since aggrecan
31 and collagen type II represent two important extra cellular matrix components of articular
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32 cartilage, which respectively constitute 95% of the total proteoglycan and 90% of the collagen
33
34 molecules (Eyre et al., 2006; Mow et al., 1992).
In addition to the chondrogenic markers, also the hypertrophic marker collagen type X was
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36 investigated in the present study. Collagen type X gene expression always attracts much attention
37
38 when hMSC chondrogenic differentiation studies are performed since hypertrophy observed in
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39 vitro, drives the neo-formed cartilaginous tissue towards endochondral ossification and bone
40 formation, rather than to a stable hyaline cartilage (Goldring et al., 2006; Mueller and Tuan,
41
2008). Therefore hMSC hypertrophic differentiation represents a major concern in cartilage
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43 tissue engineering (Goldring et al., 2006; Mueller and Tuan, 2008). Due to the hypertrophic
44 nature of the cells, mechanical loading is expected to increase the collagen type X expression
45
46 due to the stimulation of the endogenous TGF-β1 production by hBMMSCs. Indeed, collagen
47 type X gene expression it is expected to increase when TGF-β1 is present in the culture system
48 whether it be exogenous (Goldring et al., 2006; Johnstone et al., 1998) or endogenously produced
49
50
by hBMMSCs as response to mechanical loading (Li et al., 2010a). However, although
51 mechanical loading induces an increase of TGF-β1, it concomitantly enhances all the relevant
52 chondrogenic markers (collagen type II, Sox9, aggrecan) in a more physiological balance. On
53
54
the contrary, static systems that do not include TGF-β1 show reduced collagen type X expression
55 due to a lack of chondrogenic differentiation.
56 When hMwt HA was added to the culture media, it consistently reduced collagen type X gene
57
expression within the first 14 days of chondrogenesis in all three hBMMSC donors investigated.
58
59 In addition collagen X expression for the DMEM loaded group was significantly higher than
60 DMEM and HA media static groups at day 14 and 28 while the HA media loaded group was
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3 never significantly different from the static group, further demonstrating the beneficial effect of
4
5 HA media in the mitigation of collagen X expression without inhibiting the expression of the
6 chondrogenic markers collagen type II, aggrecan and Sox9 promoted by the loading conditions.
7 Previous studies carried out under static conditions and investigating the use of HA as media
8
9
supplement or as part of tissue engineered constructs described a similar reduction of collagen
10 type X expression that was also dose dependent (Amann et al., 2017; Hegewald et al., 2004;
11 Monaco et al., 2020).
12
13
14 A previous study demonstrated that eExogenous hyaluronan (MW 500-730 kDa) administered
15 by intra articular delivery, in combination with anti-inflammatory signals, have been shown to
16
actacted as disease modifying drugs showing anti-hypertrophic and pro-chondrogenic effects
17
18 (Prasadam et al., 2013). In another study, intraarticular injection of allogeneic MSCs in
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19 combination with hyaluronic acid in rabbits led to a reduction in peri-chondrocyte type X
20
collagen (Chiang et al., 2016).
21
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22 Therefore, previous studies demonstrated that mechanical load and hMwt HA, when individually
23 added to the in vitro culture systems or when HA was intra-articularly delivered in vivo, where a
24
certain mechanical loading regimen already exists, beneficially affected hBMMSC
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26 chondrogenesis and or cartilage homeostasis was beneficially affected (Chiang et al., 2016;
27 Hegewald et al., 2004; Monaco et al., 2020; Prasadam et al., 2013; Wu et al., 2017). The present
28
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study confirms the previous findings and further demonstrates that the beneficial effect on
29
30 hBMMSC chondrogenic differentiation was notably enhanced when both factors were
31 simultaneously applied to the culture environment. Indeed, the addition of hMwt HA to the
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32 culture media under mechanical loading led to an overall reduction of collagen type X gene
33
34 expression that became significant at day 14 in comparison with DMEM load. In addition
collagen X expression for the DMEM loaded group was significantly higher than DMEM and
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36 HA media static groups at day 14 and 28 while the HA media loaded group was never
37
38 significantly different from the static group, further demonstrating the beneficial effect of HA
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39 media in the mitigation of collagen X expression without inhibiting the expression of the
40 chondrogenic markers collagen type II, aggrecan and Sox9 promoted by the loading conditions.
41
The reduction of collagen type X expression in the HA media loaded group was consistent and
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43 reproducible among all three hBMMSC donors investigated especially between day 14 and 28.
44 Indeed, hMwt HA supplemented in culture media delayed and overall reduced the upregulation
45
46 of collagen X both under static and under mechanical loading conditions.
47
48 In the present study the collagen type II/X mRNA ratio, a reliable chondrocyte differentiation
49
50
marker, was also investigated. Through this ratio it is possible to gain further understanding of
51 the impact of HA media on the chondrogenic differentiation. The collagen type II/X ratio
52 improved when mechanical loading was applied to the system compared with the static
53
54
conditions (Fahy et al., 2018). However, iIn the present study, when hMwt HA was supplemented
55 into the culture media and mechanical loading was concurrently applied, an increase of collagen
56 type II and aggrecan gene expression. a notable improvement Combined with the reduced
57
collagen type X gene expression, particularly within the first 14 days of chondrogenesis, of the a
58
59 more chondrogenic collagen II/X ratio was observed at all timepoints investigated, which become
60 more marked from day 14 to day 28. Particularly at day 14 the Collagen II/X ratio was
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3 significantly higher compared with all the other groups investigated in the present study including
4
5 DMEM load. . The higher collagen II/X ratio observed in HAHA+ media loaded when compared
6 with DMEM loaded, demonstrated the beneficial effect of hMwt HA in reducing the hypertrophic
7 hMSC differentiation in favor of a more stable chondrogenesis, when associated with the loading,
8
9
in reducing the hypertrophic hMSC differentiation in favor of a more stable chondrogenesis.
10
11 In addition to collagen molecules and chondrogenic markers, lubricin gene expression was also
12 investigated in this study. Lubricin is a protein secreted by the chondrocytes mainly in the
13
14 superficial zone of native articular cartilage tissue (Jay and Waller, 2014). In a previous study
15 conducted with chondrocytes isolated from bovine fetlock joints, lubricin gene expression was
16 mainly sensitive to the mechanical loading regimen applied (Wu et al., 2017). When HA was
17 used as a media supplement at the concentration of 2mg/ml a significant increase in protein
18
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synthesis was also observed (Wu et al., 2017).
20 In the present study Lubricin (PRG4) gene expression did not show any variation within the
21 static groups, while under mechanical shear and dynamic compression a slight increase of thein
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22 gene expression was observed especially at earlier timepoints. PRG4 gene expression was
23
24
significantly upregulated under DMEM loading conditions with DMEM at day 7 compared
with the same timepoint ofthe static groups. However, when HAHA+ media was combined
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26 with loading a significant reduction of lubricin gene expression was observed over time,
27 resulting in no differences when compared with the static groups.
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The reduction reduced of the lubricin gene expression observed in the present study differs to
30 previous findings (Wu et al., 2017). However, the previous study used bovine chondrocytes
31 and only exposed the cells to HA for one hour per day. It is well known that the main function
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32 of lubricin is to provide lubrication to articulating regions and to prevent cell and protein
33
adhesion (Jay and Waller, 2014). In addition, surface motion was previously shown to be
34
specifically responsible for the lubricin level (Grad et al., 2005). Therefore, in addition to the
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36 different behavior cell type-related (chondrocytes vs hBMMSC), it is also possible that
37 hyaluronic acidHA, as a natural lubrication molecule, would reduce friction generated during
38
mechanical loading.
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40 Matrix accumulation is a combination of synthesis and degradation. In this study, the mRNA
41 expression of matrix degrading enzymes MMP3, ADAMTS4 was increased in response to
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42 mechanical load, while ADAMTS5 expression was unaffected. As the differences were similar,
43 it does not explain the enhanced safranin O staining obtained with HA addition. In addition,
44
45 HA is an ideal shock absorber and friction reducer the synovial joints due to its shear-dependent
46 viscosity (Laurent et al., 1996; Morgese et al., 2018). Therefore, if hyaluronic acid is already
47 within the media, this might reduce the needs of further hyaluronan or lubricating protein
48 production.
49
50 Previous studies demonstrate that master transcription factors, such as Sox9 for cartilage and
51 Runx2 for bone are associated with cell differentiation pathways (Bruderer et al., 2014;
52 Lefebvre and Smits, 2005). Indeed, has been also demonstrated that the propensity of hBMSCs
53 to differentiate osteogenically could be assessed through Runx2/Sox9 mRNA ratio within the
54
55
first week of osteogenic induction (Loebel et al., 2015). Therefore, the Sox9/RunX2 gene
56 expression ratio can provide information on hMSC chondrogenic differentiation.
57 In the current study, the Sox9/RunX2 chondrogenic ratio was slightly increased by the
58 mechanical loading compared with static conditions but the culture media did not affect the
59
60
ratio. This confirms the results from previous studies that included mechanical load within the
culture system (Schatti et al., 2011).
18
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