Soft Material Approach to Induce Oxidative Stress ... - ACS Publications

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A soft material approach to induce oxidative stress in mesenchymal stem cells for functional tissue repair Haibo Yang, Kim Truc Nguyen, David Tai Leong, Nguan Soon Tan, and Chor Yong Tay ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09222 • Publication Date (Web): 09 Sep 2016 Downloaded from http://pubs.acs.org on September 10, 2016

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ACS Applied Materials & Interfaces

A soft material approach to induce oxidative stress in mesenchymal stem cells for functional tissue repair Haibo Yang, †# Kim Truc Nguyen, †# David Tai Leong, ‡ Nguan Soon Tan, §∇⊥ Chor Yong Tay†§* †

School of Materials Science and Engineering, Nanyang Technological University, N4.1, 50

Nanyang Avenue, Singapore 639798, Singapore ‡

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4

Engineering Drive 4, Singapore 117585, Singapore §

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive,

Singapore 637551, Singapore ∇Institute

of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673,

Singapore ⊥

KK Research Centre, KK Women’s and Children’s Hospital, 100 Bukit Timah Road, Singapore

229899, Singapore KEYWORDS：： regenerative medicine, hydrogels, biomimetic materials, mesenchymal stem cells, reactive oxygen species

1 Environment ACS Paragon Plus


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ABSTRACT ： Biomimicking hydrogel based cell culture platforms with physiologically relevant stiffness are powerful tools to modulate the behaviors of stem cells. Herein, the use of fibronectin-conjugated polyacrylamide (PAA) hydrogel biointerface is exploited to modulate the intracellular oxidative stress of bone marrow derived human mesenchymal stem cells (MSCs). We show that compliant culture surface with kPa range matrix stiffness can augment the expression of reactive oxygen species (ROS) level in MSCs by approximately 2-4 fold compared to cells grown on conventional FN coated glass control surface in a non-cytotoxic manner. Via an unbiased proteomics approach and mechanistic studies, we show that the secretion level of a sub series of “mechano-sensitive” chemokines and trophic factors is heavily dependent on the PAA matrix stiffness mediated ROS level. Importantly, the secretome harvested from the cells that was grown on the PAA hydrogel was found to enhance wound healing in both in vitro and in vivo full thickness mouse excisional wound model. The devised “soft approach” to induce oxidative stress in MSCs is posited to pave the way for novel cell-free therapeutic interventions for the treatment of a wide variety of diseases and to foster functional tissue repair.

1. Introduction The ability to engineer stem cell interaction with the extracellular matrix (ECM) is the bedrock of biomaterials and regenerative medicine research. Stem cells interact with soluble fluid and insoluble components of the ECM to regulate a plethora of fundamental cell functions such as cell viability, proliferation, migration and differentiation.1-6 It was recently demonstrated that the inherent biochemical and biophysical cues derived from the matrix materials, in particular, substrates stiffness, nanotopography, surface chemical functionality and physical constraints, can function as pivotal cellular checkpoints, impacting the fate decisions and lineage commitment of


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stem cells via the process of mechanotransduction.1, 7-15 For instance, coerced alignment of stem cells by means of micropatterned fibronectin lanes or phage-based nanofibers and peptides can direct the differentiation of hMSCs into cells belonging to different targeted tissue lineages.1, 16-17 The diversity of these material-centric cues, thus represents a remarkable opportunity for materials scientists and stem cell biologists to co-develop advanced strategies to regulate stem cell behaviour and to discover novel materials-driven stem cell responses for regenerative medicine. Mesenchymal stem cells (MSCs) refer to a group of diverse subclass of precursor cells residing in the stromal fraction of numerous adult tissues that have been used extensively in clinical practice.18 Besides its well-established ability to differentiate into cells derived from various germ layers, MSCs also possess various unique properties such as immune-modulatory effects, self-renewability and homing ability to diseased sites.19 MSCs are also able to secrete a vast array of proteins like growth factors, cytokines, metabolites, etc, collective termed as secretome, to regulate fundamental biological processes that are crucial for tissue repair.20 Therefore, MSCs are posited to be one of the most promising cell sources for regenerative medicine, especially in the treatment of diabetes, cardiovascular diseases, neuronal disorders and wound healing.21 A steadily growing pool of evidence suggests MSCs are inherently responsive to the physical micro-environmental cues. Consequently, several engineered substrates with physiologically relevant stiffness have been exploited to modulate various aspects of MSCs behaviour, such as cell adhesion, cytoskeletal development, cellular mechanics, selfrenewability, migratory properties and even differentiation.22-25 While the action of mechanism for MSCs to translate the mechanical properties of the substrate into biochemical cues is complex, it is likely to be mediated by mechanosensitive signalling proteins such as FAK, Rho,



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YAP and α-catenin.7, 26-29 The signals from these proteins are further propagated by reactive oxygen species (ROS). ROS are highly reactive ubiquitous metabolites that play indispensable roles in signal transduction pathways to regulate several fundamental physiologic processes such as metabolism, differentiation, aging and cell death.30-31 In the context of stem cell biology, ROS can enhance self-renewal and maintain stem cell homeostasis.32-33 Despite their importance in stem cell physiology, direct effects of materialistic parameters like substrate stiffness on MSCs oxidative stress have yet to be determined. Herein, using a human plasma fibronectin (FN) conjugated polyacrylamide (PAA) hydrogel platform, we showed that matrix stiffness can function as a novel and potent regulator of the intracellular ROS level in MSCs. Compared to conventional FN coated glass substrate (GPa stiffness), MSCs cultured on the compliant PAA hydrogel with kPa range stiffness, displayed augmented level of ROS by approximately 2-4 fold, suggesting that MSCs are under certain level of oxidative stress. Interestingly, MSCs oxidatively stressed via this approach was found to be non-cytotoxic. Rather, our mechanistic studies revealed that the altered redox status of MSCs could potentially serve as a critical upstream regulator of the MSCs secretomic profile. Using a shotgun approach, several critical cytokines and growth factors such as RANTES, IL-10, OSM, L-3, IL-6, among others, were identified to be “mechanosensitive” to the substrate mechanics. More importantly, we showed for the first time that the harvested conditioned medium (containing the secretome) from MSCs cultured on the engineered PAA hydrogel could be exploited to accelerate wound healing in both in vitro and in vivo full thickness mouse excisional wound splint model.


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2. Experimental section Preparation and characterization of substrates with varying stiffness: Polyacrylamide hydrogel based substrate was prepared as described previously.7 Briefly, #1 glass coverslips (Sigma) of size 22 x 22 mm were surface treated with γ-methacryloxypropyltrimethoxysilane to render good adhesion between the polyacrylamide gel and the glass substrate. Polyacrylamide gels of differing stiffness were prepared by varying the mixing ration of acrylamide and bis acrylamide solutions. For low stiffness substrates the ratio is 4:0.15 and for intermediate stiffness substrates the ratio is 10:0.1. Polymerization of the polyacrylamide gel was initiated by addition of 10% ammonium persulfate solution (1:50 for low stiffness substrates and 1:100 for intermediate stiffness substrates) and tetramethylethylenediamine (1:5000) to the polyacrylamide solution (35 µL) that is sandwiched between the surface-treated coverslips and a transparency to attain a thin layer of polyacrylamide gel of uniform thickness. The polyacrylamide gels was first washed with 50mM HEPES buffer (pH 8.5) for 3 times and then further chemically treated with the heterobifunctional linker N-Sulfosuccinimidyl-6-[4’-azido-2’-nitrophenylaimno] hexanoate (sulfo-SANPAH) under UV (365 nm) irradiation for 15 mins to activate the gel surface that will enable crosslinking with ECM protein. After the chemical treatment, residual sulfo-SANPAH on polyacrylamide gels was removed by washing with 50 mM HEPES buffer (pH 8.5) 3 times. In this study, human plasma derived fibronectin (50 µg /ml) diluted in HEPES buffer (pH 8.5) was employed as our principal ECM protein. In each well of 6-well plate, 50 µl of aforementioned human plasma derived fibronectin solution was droped and covered by chemically treated polyacrylamide gels for cross-linking. After one hour of incubation with human plasma derived fibronectin solution in 37 ℃ incubator, the polyacrylamide gels were washed with PBS once to



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remove residual proteins and the gel is ready for use in next step. The bulk elastic moduli of the polyacrylamide hydrogels were characterized using a rheometer from Anton Paar. Cell culture and collection of MSCs conditioned medium: Only early passage ( 90%) regardless of the substrate stiffness. All data are presented as mean ± SD. (n=25) Scale bar = 100 µm. * denotes statistical significance at p 2-fold relative to the background signal) and were further quantified as shown in figure 4a. Among which, 9 of them (RANTES, IL-10, OSM, IL-3, M-CSF, TNF-α, IL-1α, MIP-1δ, SDF-1) exhibited a positive correlation with substrate stiffness, 5 of them (IL-8, MCP-1, TARC, TNF-β, VEGF) displayed a bi-modalstiffness expression profile, while IL6 was the only cytokine that showed an inverse relationship with substrate stiffness (Figure 4a). Our results suggest the existence of a subset of “mechanosensitive” secretome that are either positively or negatively correlated to substrate stiffness.



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We next attempted to validate the involvement of ROS in mediating the expression of the secreted cytokines. For this purpose, we winnowed our assessment to the four most mechanosensitive cytokine targets (i.e. IL-6, MCP-1, RANTES and IL-8), whose expression levels were shown to change significantly as a function of substrate stiffness. Specifically, IL-6 expression level correlated inversely with substrate stiffness which then peaked at the softest substrate group (1.5kPa) (Figure 4a). Conversely, MCP-1 and RANTES levels increased with increasing substrate stiffness and was the highest for MSCs cultured on the FN-coated coverslip. IL-8 exhibited a non-linear dependency on substrate stiffness. If intracellular ROS level is the stimuli for these “mechanosensitive” cytokines, then treatment of MSCs with N-acetyl-Lcysteine (NAC), a potent ROS scavenger, should attenuate the expression level of the aforementioned cytokines. Conceivably, NAC treatment would exert the same effects on the secretome expression profile, as with decreasing the ROS level in MSCs with increasing substrate stiffness would achieve. As expected, with continuous exposure of NAC (700 µM) over the incubation period, the expression level of IL-6 mRNA transcript decreased 11.6-fold when compared with the untreated group, while a concomitant upregulation in MCP-1 and RANTES mRNA transcript levels, 13-fold and 28-fold respectively, was detected. As expected, we were unable to attain a significant difference in IL-8 mRNA expression level with NAC treatment, (Figure 4b). Taken together, our results suggest that the substrate stiffness-mediated ROS level could function as a potent upstream effector to modulate the MSCs secretory cocktail (Figure 4c).


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Figure 4. Substrate stiffness modulates MSCs secretomic landscape via a ROS dependent mechanism. a) Heatmap analysis of 15 secreted proteins by MSCs revealed substrate stiffness is a potent regulator of MSCs secretome. b) The impact of NAC treatment on expression of several mechanosensitive secreted proteins was assessed at the transcript level, which is normalized to the expression level of the housekeeping 18s mRNA transcription level (dashed horizontal line). NAC treatment significantly altered expression of IL-6, MCP-1 and RANTES in a manner that is similar to culturing the cells on substrate with different stiffness, suggesting that ROS may play an important role in mediating the substrate stiffness effect. All data are presented as mean ± SD. (n=5) * denotes statistical difference at p

Soft Material Approach to Induce Oxidative Stress ... - ACS Publications

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