Sulfonated Polyrotaxane Surfaces with Basic Fibroblast Growth Factor

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Sulfonated polyrotaxane surfaces with basic fibroblast growth factor alters the osteogenic potential of human mesenchymal stem cells in short-term culture Arun Kumar Rajendran, Yoshinori Arisaka, Sachiko Iseki, and Nobuhiko Yui ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01343 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 2, 2019

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ACS Biomaterials Science & Engineering

Sulfonated polyrotaxane surfaces with basic fibroblast growth factor alters the osteogenic potential of human mesenchymal stem cells in short-term culture Arun Kumar Rajendran1‡, Yoshinori Arisaka2‡, Sachiko Iseki1, Nobuhiko Yui2*

1 Section of Molecular Craniofacial Embryology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8549, Japan.

2 Department of Organic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo, 1010062, Japan. ‡These authors contributed equally.

Corresponding author: Nobuhiko Yui, E-mail: [email protected]

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ABSTRACT

Human mesenchymal stem cells (hMSCs) are prone to senescence and lose their differentiation potential when expanded under non-favorable conditions. This leads to the underutilization of hMSCs in clinical situations such as bone regeneration. The use of growth factors and small molecules as supplements, and changing the physical properties of cell culture surface have been explored to maintain the self-renewal and differentiation potential of hMSCs during the in vitro expansion phase. Here, we have explored the effect of polyrotaxanes (PRXs) with different molecular mobilities along with either soluble or immobilized fibroblast growth factor 2 (FGF2) in the maintenance of osteogenic differentiation potential of hMSCs during in vitro expansion. We found that less expanded shape of the hMSCs was associated with highly mobile PRX surfaces and less mobile PRX surfaces led to flattened cell morphology. The presence of FGF2 induced further expansion of the cell shape and size. The immobilization of FGF2 helped to improve the yield of hMSCs on highly mobile surfaces by promoting cell attachment to the surfaces. hMSCs cultured on highly mobile PRX surfaces exhibited poor actin cytoskeletal


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organization and retention of the transcriptional regulator, yes-associated protein (YAP), in cytoplasm in contrast to the hMSCs on less mobile PRX surfaces. When the hMSCs proliferated under these conditions were collected and subjected to osteogenic differentiation on tissue culture polystyrene (TCPS) surfaces, we found that only the hMSCs cultured on highly mobile PRXs with FGF2 in both soluble and immobilized forms showed mineralization indicative of osteogenic differentiation. Further, we found that hMSCs cultured on highly mobile PRX surfaces expressed higher levels of stemness marker genes, Nanog and Oct4. These results indicate that culturing hMSCs on PRX surfaces with different molecular mobilities even for a short period of time (4 days) was sufficient to cause a drastic change in the osteogenic potential. From these results, it is suggested that apart from the use of supplements such as FGF2 in its freely soluble or immobilized form, the consideration of proper molecular mobility of the substrates could enable us to design better culture conditions for the hMSCs with osteogenic potential.

Keywords: polyrotaxane, fibroblast growth factor, molecular mobility, surfaceimmobilization, mesenchymal stem cell.


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INTRODUCTION

Stem cells are of great therapeutic value in the field of regenerative medicine and tissue engineering1. Mesenchymal stem cells (MSCs) are used for regeneration in bone defects because they are readily differentiated into osteoblasts2-4. The MSCs isolated from the patient are expanded in vitro and delivered at the required site with help of carriers such as hydrogels and scaffolds5-7. Although the protocols for successful and easy isolation of human MSCs (hMSCs) have been greatly improved along with numerous carrier systems that enable successful transfer of hMSCs into the defect site, one of the major problems encountered is loss of differentiation potential during the in

vitro expansion phase8. Isolated MSCs can enter into senescence or lose their differentiation potential under the inappropriate culture conditions including long culture period more than 100 days, during the expansion phase9-11. This undesired effect can be overcome by choosing the appropriate type of cell growth medium with addition of certain supplements such as proteins/peptides and small molecules in the cell culture


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medium as well as type of cell culture surfaces with physical or chemical modifications8, 12-14.

It is known that the mechanical/physical properties of cell culture surfaces play a major role in most of the cellular responses including proliferation, self-renewal, apoptosis and differentiation13, 15. Previous researches have shown that the self-renewal and differentiation ability of MSCs could be regulated through mechanosensing pathways by modifying the physical properties of the cell culture surfaces with their topography and roughness, and introducing features such as nano/micropillars, hardness, elasticity, and molecular mobility15-16. Moreover, hMSCs can be made quiescent rather than senescent, when grown on soft substrates17.

Polyrotaxane (PRX) is a supermolecule which consists of an axle polymer such as poly(ethylene glycol) (PEG) onto which cyclic molecules such as α-cyclodextrins (CDs) are initiatively threaded. Upon hydration or stimulation, it is expected that the CDs slide or rotate along the axle polymer. This movement is referred to as molecular mobility. We have previously shown that the extent of molecular mobility can be controlled by


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changing the number of threading CDs onto the PEG chain16,18. Furthermore, molecular mobility can influence numerous signaling pathways by altering the status of cytoskeleton, e.g. organization of actin filaments (F-actin), RhoA-ROCK activity, and Rac1 expression among others16,19,20. These pathways have been shown to be involved in the maintenance of hMSC quiescence and self-renewal21-22. By altering the cytoskeletal signaling pathways, the commitment of stem cells can be greatly influenced, as shown in our previous reports16,20. Since mechanosignalling are relatively quick events, we hypothesized that different molecular mobilities could affect the stemness or the differentiation potential of hMSCs even during their expansion phase. In addition, sulfonation of the CDs in the PRXs could mimic the endogenous sulfonated proteoglycans such as heparin and heparin sulfate, which are well known to enhance cell survival23.

Fibroblast growth factor 2 (FGF2) is a class of heparin-binding growth factor that is known to be one of the key proteins for self-renewal and maintenance of differentiation capability of hMSCs24. This has been attributed to the ability of FGF2 to activate the


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PI3-Akt, ERK1/2, and JNK signaling pathways in hMSCs25-26. Upon the usage of FGF2 as a key component for the self-renewal and maintenance of hMSCs, researchers have tried to improve the efficiency of FGF2 through various methods of application, such as regulated delivery of FGFs and immobilizing FGFs onto the cell culture surfaces rather than the conventional addition of FGFs in cell culture media in freely soluble form. From previous reports, immobilizing FGFs onto cell culture surfaces seems plausible owing to the ease of immobilization and reduction in the dose needed27. Therefore, immobilization of FGF2 onto the cell culture surfaces could be of great advantage in hMSC cultures.

In this study, we utilized PRXs that were modified with sulfopropyl ether groups (SPEPRX) to analyze how the change in the number of threading CDs in SPE-PRX alters hMSC response and osteogenic differentiation potential during the expansion phase of culture. Furthermore, as FGF2 is one of the key factors for maintenance of multipotency of hMSCs, we also attempted to elucidate the effect of freely soluble FGF2 in the culture media and immobilized FGF2 over SPE-PRX surfaces. This study could help us gain


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some insights into the favorable combination of SPE-PRX surfaces and the method of FGF2 application to maintain osteogenic differentiation during the expansion phase of hMSCs with considerable yields. The insights provided by this study could help us design surfaces for stem cell expansion for specific clinical purposes in the future.

EXPERIMENTAL SECTION

MATERIALS

Sulfonated-PRX triblock copolymers composed of sulfopropyl ether-modified α-CDs threaded onto a PEG chain (Mn = 20,000) as a middle PRX segment and poly(benzyl methacrylate) (PBzMA) at both terminals of the PEG as anchoring segments (SPEPRXs), as well as unsulfonated PRX triblock copolymers, were prepared as described previously28-29. SPE-PRXs with different numbers of threading CDs were obtained by altering the PEG/α-CD molar ratios. SPE-PRX surfaces were coded as “SPE-PRXX,” where X indicates the number of threading α-CD. hMSC were obtained from Lonza


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(Walkersville, MD, USA). Recombinant human FGF2 was purchased from Pepro Tech (Rocky Hill, NJ, USA). Paraformaldehyde (4%) was purchased from Wako Pure Chemicals (Osaka, Japan). Dimethyl sulfoxide (DMSO) was purchased from Kanto Chem. (Tokyo, Japan). Twenty-four-well tissue culture polystyrene plates were purchased from Thermo Scientific (Rockford, IL, USA).

METHODS

Fabrication of SPE-PRX surfaces and immobilization of FGF2

SPE-PRX was dissolved in DMSO at 0.05 w%. SPE-PRX was coated onto the 24-well tissue culture polystyrene plates by using the simple drop casting method. Then, 30 µL of SPE-PRX5 or SPE-PRX86 was drop-casted and allowed to dry in the drying oven overnight. After confirming the complete drying of SPE-PRX, the well plates were washed three times with Dulbecco's phosphate buffered saline without calcium and magnesium (PBS) and sterilized using ultra-violet illumination in a cell culture bench for 30 min. The values of zeta potentials on SPE-PRX5 and SPE-PRX86 were -27.2 ± 2.1 and -48.3 ± 3.3, respectively29.In order to immobilize FGF2 onto SPE-PRX surfaces,


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500 μL of FGF2 solution (0.04 ng/μL) was added to each surface. These were then incubated at 37°C for 6 h, resulting in SPE-PRX surfaces with immobilized FGF2 (SPEPRX/Immo-FGF2).

In vitro cell culture

hMSCs were expanded on tissue culture polystyrene plates using mesenchymal stem cell media obtained from PromoCell (Heidelberg, Germany). Fifth-passaged cells were used for this study. Briefly, expanded hMSCs were trypsinized, collected and seeded in the wells, at a density of 1.6×104 cells/well and cultured under three different conditions of FGF2 application; without FGF2 (No FGF2), with soluble FGF2 (Sol. FGF2) or with immobilized FGF2 (Immo. FGF2), as shown in Figure 1. For the SPE-PRX/Sol. FGF2 surfaces, 20 ng of FGF2 was added to the cell culture media at 1 h post-cell seeding. These cells were allowed to proliferate for 3 days and on the fourth day, the cells were trypsinized, collected, and counted using a cell counting chamber. Then, cells from each group were seeded onto normal 24-well plates at a density of 1×104 cells/well and cultured using a normal growth medium. At a pre-confluence state, on the fourth day,


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the growth medium was replaced with an osteogenic induction medium (Mesenchymal Stem Cell Osteogenic Differentiation Medium, Promocell, Germany).

Assessment of cell density at different time points

After seeding the hMSCs onto different SPE-PRX surfaces, bright field images were acquired at 24-h intervals for 3 days using a phase contrast microscope (IX71; Olympus, Tokyo, Japan) equipped with a dual CCD digital camera (DP80; Olympus). The number of cells was calculated manually by ImageJ using 8 randomly chosen fields per group (NIH, Bethesda, USA), and cell-counting chamber (OneCell, Tokyo, Japan) after the trypsinization of cells.

Cellular spreading area measurements

The cellular spreading area was calculated using ImageJ. The scale was calibrated using a bright field image of a cell counting chamber at the same magnification. The perimeter of the cells was manually traced, and the area values were obtained using the measure tool. At least 40 cells in each group were analyzed.


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Actin and YAP staining and analysis

Actin organization in hMSCs over different SPE-PRX surfaces were analyzed 24 and 96 h post seeding. After 24 or 96 h of culture, the cells were fixed with 4% paraformaldehyde/PBS (PFA) for 10 min, following which the cells were permeabilized using 0.1% digitonin/PBS for 5 min at room temperature. Non-specific blocking was carried out by incubating the cells with 3% bovine serum albumin solution in PBS for 1 h at room temperature. Primary yes-associated protein (YAP) antibody (Cell Signaling Technology, Danvers, MA, USA) was added in 1:1000 dilution in PBS to the cells and incubated at 4 °C overnight followed by incubation in dark with Alexa flour 488® conjugated IgG secondary antibody (Abcam, Cambridge, UK) at 1:2000 in PBS for 1 h. Cells were then stained with Alexa Flour 555® conjugated Phalloidin (Invitrogen, Carlsbad, CA, USA), at 1:100 dilutions in PBS for 20 min followed by 1:500 diluted Hoecsht 33342 (Dojindo, Kumamoto, Japan) in PBS for 15 min at room temperature. PBS was used to wash the cells in the intermediate and final steps. The stained cells were imaged using appropriate fluorescent filters equipped phase contrast microscope (IX71;


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Olympus, Tokyo, Japan) equipped with a dual CCD digital camera (DP80; Olympus). A minimum of 6 random fields were imaged per condition, and the images were analyzed using ImageJ. hMSC stemness gene expression analysis

The short-term effect of molecular mobility on the stemness potential of hMSCs was analyzed by confirming the expression of stemness markers using quantitative PCR. Briefly, hMSCs were cultured and proliferated on highly and less mobile SPE-PRX surfaces for 3 days. On 4th day, the total RNA was isolated from the cells by using a FastGeneTM RNA Premium Kit (NIPPON Genetics, Tokyo, Japan) according to the manufacturer’s protocol and the concentration was measured using Biospec-Nano (Shimadzu Biotech, Japan). Equal quantities of mRNA from each sample was used for reverse transcription using a ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). The expression levels of CD44, Nanog and Oct4 relative to Gapdh were analyzed by quantitative PCR using THUNDERBIRD SYBR qPCR Mix (Toyobo) in a


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CFX connect real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The primer sequences used for PCR are given in the supplementary Table S1.

Estimation of osteogenic differentiation

After two weeks of culturing hMSCs in osteogenic differentiation medium (Lonza), the media was removed, and the wells were washed using PBS. Then, the cells were fixed with 4% PFA by incubating for 10 min, after which they were washed with Milli-Q water. Then, 250 µL of 1% alizarin red S (ARS) solution in Milli-Q was added and incubated. After 10 min, the ARS solution was removed and the wells were washed with Milli-Q water. The cells were imaged under bright field using a phase contrast microscope (IX71; Olympus, Tokyo, Japan). After imaging, the well plates were allowed to air dry. After drying, 500 µL of DMSO was added to each well (n=3) and kept under mild shaking for 30 min to completely elute the ARS. Then, 100 µL aliquots of DMSO containing ARS from these wells were obtained and absorbance was measured at 405 nm using a spectrophotometer (JASCO, Tokyo, Japan).

Statistical analysis


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Numerical results are expressed as the average ± standard deviation (S.D.). Analysis of variance (ANOVA) followed by Tukey’s posthoc test was applied for finding statistical differences between the groups. The p-value was set at 0.05 for statistical significance.

RESULTS AND DISCUSSION

Molecular mobility and application of FGF2 affects cellular shape and spreading area

hMSCs were seeded onto the surface either SPE-PRX5 with highly molecular mobility or SPE-PRX86 with less molecular mobility under three different FGF2 conditions; No FGF2, Sol. FGF2 and Immo. FGF2. Bright field images were acquired and analyzed for measurement of cellular shape and size to compare early and late cellular responses, at 24 h (Supplementary Figure S1) and 72 h culture after seeding, respectively (Figure 2A). It was evident that the cells on SPE-PRX5/No FGF2 surfaces were mostly rounded in shape and small in size, even after 72 h. However, cellular adhesion on SPEPRX5/Sol. FGF2 and SPE-PRX5/Immo. FGF2 surfaces seemed to be improved after 72


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h compared to that after 24 h at which the cellular shape was rounded. In contrast, SPE-PRX86/No FGF2 surfaces indicated attachment of the cells to some extent with any FGF2 conditions at 24 h although better attachment was observed in the presence of FGF2. After 72 h, the SPE-PRX86/Sol. FGF2 and SPE-PRX86/Immo. FGF2 surfaces showed higher levels of cellular spreading compared to SPE-PRX86/No FGF2 surface, and it was clear that the presence of FGF2 induced active cellular proliferation. This is consistent with our previous observations that the cells on highly mobile surfaces tend to show round and lesser spreading characteristics and cellular spreading was greatly enhanced on less mobile PRX surfaces25.


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Figure 1. Chemical formula of synthesized SPE-PRX and schematic illustration of hMSCs culture in this study. hMSCs are cultured on SPE-PRX surfaces with or without FGF2 in a growth medium for 3days. On 4th day, the cells are collected and seeded onto tissue culture polystyrene surfaces using regular growth medium. After culturing for another 3 days, the medium was changed to osteogenic induction medium, followed by culturing for 14 days.


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Figure 2. Phase contrast microscopic images (A) and cellular spreading areas (B) of hMSCs cultured on SPE-PRX surfaces with different numbers of α-cyclodextrin threading. hMSCs were cultured on the surfaces for 72 h under three different FGF2 conditions; No FGF2, Sol. FGF2 and Immo. FGF2. The data are expressed as the mean ± S.D. of triplicate experiments. Statistical significance was determined by analysis of variance followed by Tukey's test. * represents p < 0.05. Scale bar represents 250 µm.


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The quantitative analysis of cellular spreading area at 72 h (Figure 2B) showed that the mean area of cells on SPE-PRX5/No FGF2 surfaces was 984.5 ± 693.2 µm2, whereas those on the SPE-PRX5/Sol. FGF2 and SPE-PRX5/Immo. FGF2 surfaces were 1690.4 ± 1074.4 and 2169.1 ± 1484.4 µm2, respectively. The areas with FGF2 became significantly higher than those without FGF2. hMSCs cultured on SPE-PRX86/No FGF2, SPE-PRX86/Sol. FGF2, and SPE-PRX86/Immo. FGF2 surfaces showed areas of 2834.2 ± 1840.1, 5413.6 ± 3232.0, and 5424.9 ± 3257.3 µm2, respectively. SPE-PRX86 surfaces exhibited significantly higher levels of cellular spreading area than SPE-PRX5 surfaces. Even among the SPE-PRX86 surfaces, the addition of FGF2 increased the flattering of cells. These results indicate that the presence of FGF2 is necessary for enhancing cellular attachment and spreading. It is known that round cell morphology similar to that on SPE-PRX5/No FGF2 surfaces causes apoptosis to adherent cells such as hMSCs, as these cells can sense that the microenvironment is not suitable30. Further, previous studies demonstrated that excessive cellular spreading was also indication of the senescence of cells, suggesting that the modulation of cellular spreading could play important roles in regulation of hMSC differentiation potential30-32.


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Furthermore, researchers have demonstrated that lowering the cell-matrix tractions, which reduces cellular spreading could maintain the plasticity of stem cells33.

Molecular mobility and mode of FGF2 application affects hMSC yield

The number of hMSCs per well was calculated at 24 and 96 h in culture (Figure 3). The number of yielded cells at 96 h was about as two times as that of 24 h for all conditions. The SPE-PRX86 surfaces resulted in significantly higher number of hMSCs than the SPE-PRX5 surfaces at both time points. This could be because highly mobile PRX-surfaces could exhibit mechanics similar to soft extracellular matrices, which is not favorable for hMSC adhesion, whereas less mobile PRX-surfaces may be similar to a hard matrix to which cells can adhere well, spread and increase in number. However, it was evident that the Immo. FGF2 condition showed significantly higher number of hMSCs than other FGF2 conditions for both SPE-PRX5 and SPE-PRX86 surfaces. This is consistent with our previous studies and other studies, which showed that immobilization of proteins, such as bone morphogenetic protein 2, epidermal growth factor etc., can enhance cell yield29,34. This could be due to the fact that immobilized


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peptides aid in the better adhesion of cells on to the surfaces rather than directly stimulate cellular proliferation. Furthermore, surface-immobilized growth factors are more accessible to the cells compared to those dissolved in the solution, thereby leading to continuous and enhanced downstream signaling34. These results clearly indicate that the immobilization of FGF2 to SPE-PRX surfaces is important to increase the yield of hMSCs.

Figure 3. Number of adhering hMSCs on SPE-PRX surfaces with different numbers of threading α-cyclodextrins. hMSCs were cultured on the surfaces for 24 h (A) and 96 h (B), under three different FGF2 conditions; No FGF2, Sol. FGF2 and Immo. FGF2. The data are expressed as the mean ± S.D. of triplicate experiments. Statistical significance was determined by analysis of variance followed by Tukey's test. * represents p < 0.05.


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Actin cytoskeletal organization is altered by molecular mobility in hMSCs

The organization of one of the key cytoskeletal structures, F-actin, in hMSCs was analyzed by staining with Alexa Flour 555® conjugated Phalloidin. After 24 and 96 h of culture over different molecular mobility surfaces under different conditions, we could find that SPE-PRX5 surfaces showed poorly organized F-actin filaments when compared to extensive and well-organized F-actin filaments in cells grown on SPE-PRX86 surfaces (Figure S2 (A), Figure 4 (A)). Since culturing on PRX0 surfaces with non-threading αCDs prevented cells from attaching onto the surfaces and easily removed during media changes or washing steps (data not shown and from our previous experiences), these differences are expected to be due to the presence of α-CDs. Immobilization of FGF2 on SPE-PRX5 seemed to slightly increase the F-actin organization, indicating that adhesive surface area was higher than No FGF2 or Sol. FGF2 condition. The wellorganized and clear actin filaments, as seen in the cells grown on less molecular mobile surfaces, are indicative of high cytoskeletal tension. The key role of actin in mechanosensing of the microenvironment is well known. Douglas Zhang et al. have


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noticed that culturing MSCs in small micro islands reduced spreading and the cytoskeletal tension. This in turn made the MSCs to become quiescent, confirmed by the higher expression of stemness markers35. Consistently in our experiments, the highly mobile PRX surfaces showed decreased spreading of hMSCs, weak and poor organization of actin microfilaments, which suggests hMSC quiescence due to substrate’s mechanosignalling.


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Figure 4. Fluorescence microscopy images of adhering hMSCs on the surfaces of SPE-PRX5 and SPE-PRX86, under three different FGF2 conditions; No FGF2, Sol. FGF2 and Immo. FGF2 (A). After 96-h cultivation at 37°C, cell nuclei (blue), YAPs (green) and F-actin (red) in hMSC were stained. Scale bars: 100 μm. Proportion of nuclear YAP localization (filled bars), both nuclear and cytoplasmic YAP localization (hatched bars) or cytoplasmic YAP (opened bars) in hMSCs on each surface (B) n ≥ 60.


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Molecular mobility determines the YAP compartmentalization

The immuno-fluorescent images of YAP staining revealed that, at the end of 24 h, almost all the hMSCs, on highly mobile PRX surfaces with FGF2 did not exhibit any nuclear compartmentalization of YAP; rather higher percentage of cells expressed YAP equally in cytoplasm and nucleus (Figure S2). However, SPE-PRX5/No FGF2 surface showed an increase in percentage of hMSCs expressing nuclear YAP compared to SPE-PRX5/Sol. FGF2 and Immo. FGF2 surfaces. Strikingly, all the SPE-PRX86 surfaces exhibited higher percentage of cells with nuclear YAP localization just after 24 h of culture compared to SPE-PRX5. This was clearly indicative of vast differences in the mechanosignalling machinery due to the molecular mobility of PRXs could affect over a very short period. Furthermore, at the end of pre-culture period it was evident that, higher percentage of hMSCs on highly mobile PRX surface showed YAP in the cytoplasm, except for SPE-PRX5/No FGF2 surface (Figure 4). In contrast, most of the hMSCs cultured over less mobile PRX surface exhibited YAP predominantly in the


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nucleus. YAP, one of the key mechanosignalling molecules, also functions as a transcriptional regulator of various genes depending on its localization36. Various researchers have shown that the active phosphorylated YAP can be formed due to the spreading of cells and increase in the cytoskeletal tension. This active YAP can translocate into the nucleus along with proteins like TAZ, TEAD, SMADs and can prime the cells for differentiation into specific lineages36. In our study, we found that predominant number of hMSCs retained the YAP in the cytoplasm, non-activated YAP, when cultured over the highly mobile PRX surfaces. We consider that such inactivated form for YAP could help in maintaining the hMSCs’ quiescence and differentiation potential.

Molecular mobility and mode of FGF2 application affects the osteogenic differentiation potential of hMSCs

The hMSCs expanded on different SPE-PRX surfaces with different FGF2 conditions for four days were subjected to osteogenic induction on TCPS surfaces to investigate the effect of different culture conditions during the expansion phase of hMSCs on cell


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potency to differentiate into osteoblasts. After two weeks in osteogenic induction media followed by ARS staining, only the hMSCs expanded over SPE-PRX5/Sol. FGF2 and SPE-PRX5/Immo. FGF2 surfaces showed extensive mineralization (Figure 5), which was confirmed by the ARS quantification. Of contrast, the hMSCs expanded on SPEPRX86 surfaces failed to form noticeable mineralization nodules regardless of the presence of FGF2. These results clearly indicated that hMSCs could lose their osteogenic differentiation capability when cultured on less mobile surfaces, but could maintain the potency when cultured on highly mobile SPE-PRX surfaces. However, even on highly mobile SPE-PRX surfaces, the application of FGF2 was necessary for maintaining the maximum differentiation capability of hMSCs. These finding suggest that osteogenic differentiation ability of hMSCs can be altered during the expansion phase. It has been shown that culturing hMSCs for extended periods of time can lead to senescence9-10. However, in our study, we observed that cell-cultivation on SPE-PRX surfaces with less mobilities for only 4 days demonstrated a significant effect on differentiation potency of hMSCs into osteoblasts. This indicates that mechanical stimuli over a short time span could be enough to control cell fate. Consistently, Yang et al.


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reported that hMSCs can exhibit memory from the mechanical stimuli of the substrates and that the dose of these mechanical stimuli can greatly influence the fate of hMSCs37.

Figure 5. Osteogenic differentiation of human mesenchymal stem cells. After hMSCs were cultured on SPE-PRX surfaces for 4 days under three different FGF2 conditions; No FGF2, Sol. FGF2 and Immo. FGF2, and the cells were cultured on TCPS surfaces in normal growth medium for 3 days, then in an osteogenic induction medium for 2 weeks. After inducing osteogenic differentiation, the cells were stained with alizarin red S (A). Quantification of the alizarin red S stain was performed by eluting the dye using dimethyl sulfoxide and measuring the optical density


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at 405 nm (B). The data are expressed as the mean ± S.D. of triplicate experiments. Statistical significance was determined by analysis of variance followed by Tukey's test. Scale bar represents 400 µm.

Highly molecular mobility surface maintains the hMSCs quiescence

In order to confirm whether mechanosignalling using molecular mobility of SPE-PRX could alter the hMSC quiescence after a short duration of preculture, the hMSC marker genes CD44, Nanog, and Oct4 were analyzed for hMSCs expanded on SPE-PRX5 and SPE-PRX86 surfaces (Supplementary Figure S3). We found that the relative gene expression levels of Nanog and Oct4 were reduced in the hMSCs cultured over SPEPRX86 surface. However, the expression level of CD44 remained the same. This indicates that the mechanical stimuli such as molecular mobility from the substrates could play a vital role in changing the key cellular functions even in a short duration of time. This is consistent with other previous researches which have shown that various mechanical properties of cell culture substrates could alter the quiescent state of MSCs32,33,37.


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CONCLUSIONS

This study explored the effect of molecular mobility of SPE-PRX surfaces in altering the osteogenic differentiation potential of hMSCs during their expansion phase. Our results indicate that hMSCs on highly mobile SPE-PRX5 surfaces, exhibiting less adhesion, were prevented from entering senescence or losing their osteogenic differentiation potential. Notably, the yield of hMSCs was compensated by immobilizing FGF2 to highly mobile surfaces, although not up to the level of less mobile surfaces. Furthermore, there was notable difference in the mechanosignalling elements, such as actin fiber organization and YAP compartmentalization, due to different molecular mobilities. Therefore, the advantage of using highly mobile SPE-PRX5 surfaces with immobilized FGF2 for hMSC expansion was evident when osteogenic differentiation after the expansion is induced. Although this study evaluated only the short-term expansion of hMSCs on different substrates, we noted that hMSCs significantly lost their osteogenic differentiation capacity on less mobile surfaces in such a short duration. In future, this system could be modified and customized by fine-tuning a variety of


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factors, including the number of threading CDs and the concentration of FGF2 used for immobilization, to increase the yield without compromising hMSC differentiation potentials including adipocyte and chondrocyte. It could be also useful for designing surfaces for expansion of other stem cells for specific clinical applications.

Author Contributions The manuscript was written through contributions of all authors.

‡These authors contributed equally.

Funding Sources This work was supported by the Grant-in-Aid for Encouragement of Young Scientists (B) from Japan Society for the Promotion of Science (JSPS) (No. 16K16399 to Y.A.); the Grant-in-Aid for Scientific Research (A) from JSPS (No. 16H01852 to N.Y.); Interdisciplinary and International Project for Development of Advanced Life-innovative


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Materials and Human Resources grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT); the Cooperative Project among Medicine, Dentistry, and Engineering for Medical Innovation grant from MEXT; the Azuma Medical & Dental Research Grant [to Y.A.].

Supporting information

Primer sequences for quantitative real‑time PCR, phase contrast microscopic images of hMSCs after a 24-h culture, fluorescence microscopy images of YAP in MSCs, stemness gene expressions in hMSCs

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For Table of Contents Use Only Title: Sulfonated polyrotaxane surfaces with basic fibroblast growth factor alters the osteogenic potential of human mesenchymal stem cells in short-term culture Author: Arun Kumar Rajendran1‡, Yoshinori Arisaka2‡, Sachiko Iseki1, Nobuhiko Yui2* 1 Section of Molecular Craniofacial Embryology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8549, Japan. 2 Department of Organic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo, 101-0062, Japan. ‡These authors contributed equally.


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Sulfonated Polyrotaxane Surfaces with Basic Fibroblast Growth Factor

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