Substrate stiffness- and topography-dependent differentiation of annulus fibrosus-derived stem cells is regulated by Yes-associated protein

Acta Biomaterialia 92 (2019) 254–264

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Substrate stiffness- and topography-dependent differentiation of annulus fibrosus-derived stem cells is regulated by Yes-associated protein Genglei Chu a,1, Zhangqin Yuan a,1, Caihong Zhu a,1, Pinghui Zhou b,1, Huan Wang a, Weidong Zhang a, Yan Cai a, Xuesong Zhu a, Huilin Yang a, Bin Li a,c,⇑ a

Department of Orthopaedic Surgery, Orthopaedic Institute, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China Department of Orthopaedic Surgery, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui, China c China Orthopaedic Regenerative Medicine Group (CORMed), Hangzhou, Zhejiang, China b

a r t i c l e

i n f o

Article history: Received 20 December 2018 Received in revised form 24 April 2019 Accepted 6 May 2019 Available online 9 May 2019 Keywords: Annulus fibrosus Annulus fibrosus-derived stem cells YAP Poly(ether carbonate urethane)urea Stiffness Topography Differentiation

a b s t r a c t Annulus fibrosus (AF) tissue engineering has attracted increasing attention as a promising therapy for degenerative disc disease (DDD). However, regeneration of AF still faces many challenges due to the tremendous complexity of this tissue and lack of in-depth understanding of the structure-function relationship at cellular level within AF is highly required. In light of the fact that AF is composed of various types of cells and has gradient mechanical, topographical and biochemical features along the radial direction. In this study, we aimed to achieve directed differentiation of AF-derived stem cells (AFSCs) by mimicking the mechanical and topographical features of native AF tissue. AFSCs were cultured on four types of electrospun poly(ether carbonate urethane)urea (PECUU) scaffolds with various stiffness and fiber size (soft, small size; stiff, small size; soft, large size and stiff, large size). The results show that with constant fiber size, the expression level of the outer AF (oAF) phenotypic marker genes in AFSCs increased with the scaffold stiffness, while that of inner AF (iAF) phenotypic marker genes showed an opposite trend. When scaffold stiffness was fixed, the expression of oAF phenotypic marker genes in AFSCs increased with fiber size. While the expression of iAF phenotypic marker genes decreased. Such substrate stiffness- and topography-dependent changes of AFSCs was in accordance with the genetic and biochemical distribution of AF tissue from the inner to outer regions. Further, we found that the Yes-associated protein (YAP) was translocated to the nucleus in AFSCs cultured with increasing stiffness and fiber size of scaffolds, yet it remained mostly phosphorylated and cytosolic in cells on soft scaffolds with small fiber size. Inhibition of YAP down-regulated the expression of tendon/ligament-related genes, whereas expression of the cartilage-related genes was upregulated. The results illustrate that matrix stiffness is a potent regulator of AFSC differentiation. Moreover, we reveal that fiber size of scaffolds induced changes in cell adhesions and determined cell shape, spreading area, and extracellular matrix expression. In all, both mechanical property and topography features of scaffolds regulate AFSC differentiation, possibly through a YAP-dependent mechanotransduction mechanism. Statement of Significance Physical cues such as mechanical properties, topographical and geometrical features were shown to profoundly impact the growth and differentiation of cultured stem cells. Previously, we have found that the differentiation of annulus fibrosus-derived stem cells (AFSCs) could be regulated by the stiffness of scaffold. In this study, we fabricated four types of poly(ether carbonate urethane)urea (PECUU) scaffolds with controlled stiffness and fiber size to explore the potential of induced differentiation of AFSCs. We found that AFSCs are able to present different gene expression patterns simply as a result of the stiffness and fiber size of scaffold material. This work has, for the first time, demonstrated that larger-sized and higher-stiffness substrates increase the amount of vinculin assembly and activate YAP signaling in

⇑ Corresponding author at: 708 Renmin Rd, Rm 308 Bldg 1, Soochow University (South Campus), Suzhou, Jiangsu 215007, China. 1

E-mail address: [email protected] (B. Li). These authors contribute equally to this work.

https://doi.org/10.1016/j.actbio.2019.05.013 1742-7061/Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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pre-differentiated AFSCs. The present study affords an in-depth comprehension of materiobiology, and be helpful for explain the mechanism of YAP mechanosensing in AF in response to biophysical effects of materials. Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Annulus fibrosus (AF) is a multi-lamellar fibrocartilage tissue that forms intervertebral disc (IVD) together with the nucleus pulposus (NP). As a crucial supporting component of IVD, AF is required to maintain its structural and mechanical integrity to retain NP, allowing disc compression/decompression [1]. Injuries in AF tissue can lead to significant remodeling and deterioration of the entire IVD, resulting in degenerative disc disease (DDD) and lower back pain. Current clinical treatments are primarily palliative, and strategies based on tissue engineering (TE) are dramatically needed which are expected to restore the physiologic function of IVD and alleviate lower back pain [2]. Numerous strategies were proposed to engineer AF tissue. However, no engineered AF tissue has yet entered clinical practice because few of these engineered tissues fully replicate the heterogeneous mechanical property and angle-ply microstructural characteristics of their native counterparts [3–5]. In AF, significant difference exists between various regions along the radial direction with respect to cellular phenotype, matrix composition, structural characteristics, and mechanical properties [6]. Ligament-like tissues are dominant in the outer AF (oAF), whereas cartilage-like tissues are more abundant in the inner AF (iAF) [7,8]. The content of type I collagen (Col-I) is higher and the collagen fibrils are thicker and more tightly packed in oAF region than iAF, which collectively contribute to the higher stiffness of oAF. In contrast, iAF contains more Aggrecan and type II collagen (Col-II) with thinner fibril and larger extrafibrillar spacing [9–11]. Moreover, it was reported that the AF phenotypic markers Adamts17, Col5a1, Col12a1 and Sfrp2 were more substantially expressed in the oAF compared with iAF tissue in human and bovine IVDs [12,13]. Collagen fibers are major components of extra cellular matrix (ECM), and their features play determining role in adjusting cell-ECM adhesion, affecting the cell spreading area, regulating cell differentiation and determining cell stemness maintenance [14]. In a similar way, the biophysical cues, like mechanical, topographical, and geometrical features, of engineered biomaterials can also influence cell behaviors and functions significantly [15,16]. Therefore, effective replication of the region-specific features of AF is critical for fabricating scaffolds for AF TE. To date, a number of studies have attempted to replicate the heterogeneous characteristics of AF tissue. Lamellar scaffolds with oriented silk fibers was reported to direct alignment of human chondrocytes and deposited ECM, which could be used for AF TE [17,18]. Our previous studies have also demonstrated that the fiber orientation and stiffness of electrospun scaffolds affected gene expression in annulus fibrosus-derived stem cells (AFSCs), which possess the potential to differentiate into all types of resident cells in native AF tissue [19–21]. Recently, physical cues such as physical properties, topographical and geometrical features were shown to profoundly impact the growth and differentiation of cultured stem cells. For instance, stiff substrates accelerate osteogenic differentiation, which can be further promoted by the introduction of disordered nanopatterns [22,23]. Soft substrates with an ordered nanotopography increase neuronal differentiation even in the absence of induction factors [24,25]. Until now, most reports regarding the impacts of materials on cell functions have been case-by-case studies evaluating

discrete phenomena [26]. Systematic investigation of the mechanisms by which material properties affect cell functions to decide cell fate and tissue/organ development is still lacking [27]. Given the in vivo relation between the biophysical characteristics of fibrils and ECM deposition within AF tissues, it is reasonable to hypothesize that variation in mechanical and topographical features may synchronously influence the behaviors of AF cells including AFSCs. While several studies have reported cellular response to engineered mechanical and topographical signals, the underlying mechanism of AFSCs’ response to mechanical and topographical signal remains to be discovered [27]. The capacities of cells to perceive mechanics and structures of ECM are attributed to the Hippo pathway effector Yes-associated protein (YAP) and its transcriptional co-activator PDZ-binding motif (TAZ). These proteins shuttle to the nucleus to exert cotranscriptional activity [28]. In mechanical conditions in which cells develop low contractile forces (for example, when they are grown on small adhesive areas favoring a small cell size, on soft ECM or on top of bendable micropillars), YAP is in an inactivated phosphorylated form and localized at the cytoplasm. However, it is dephosphorylated and relocated to the nucleus and becomes active under experimental mechanical conditions that favor the development of high intracellular resisting forces [29]. After binding to cell- and context-specific transcription factors, YAP can regulate gene expression and thereby remodel ECM [30]. In bone marrow mesenchymal stem cells (MSCs), active expression of nuclear-active YAP was found to promote osteogenic differentiation while low YAP activity led to adipogenesis [31,32]. Regulation of activity and distribution of YAP was found in cells cultured on micropatterned substrates with the same stiffness but different structures, and the cells showed different degrees of spreading associated with YAP activation [33]. Recent findings showed that YAP also negatively regulated chondrogenesis in response to mechanical cues [34]. He et al. also reported that YAP regulates periodontal ligament cell differentiation into myofibroblast interacted with RhoA/ROCK pathway [35]. In all, mechanosensitive cell differentiation might be regulated by the mechanosensitive transcriptional regulator YAP [30]. However, the functional relation between the biophysical cues and YAP activity in AF cells remains unclear. Toward this aim, we fabricated a series of aligned fibrous scaffolds with various stiffness and fiber size using electrospinning method to mimic the heterogeneous characteristics of native AF tissue. We investigated the effects of stiffness and fiber size of scaffolds on AFSC behaviors. Further, we examined the connection between such regulation and the YAP pathway, and identified the relationship between YAP and FA-cytoskeleton remodeling in mechanical- and topography-driven differentiation of AFSCs. 2. Experimental section 2.1. Fabrication of electrospun fibrous PECUU scaffolds PECUU scaffolds were fabricated using electrospinning technique and the stiffness of the scaffolds was measured using nano-indentation test according to our previously reported methods [10,21]. In brief, PECUUs with high and low stiffness was dissolved in hexafluoroisopropanol to prepare 12 and 24 wt% for

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fabricating small and large diameter fibers respectively. As a result, four types of poly(ether carbonate urethane)urea (PECUU) scaffolds with controlled stiffness and fiber size: soft, small diameter (SS); stiff, small diameter (FS); soft, large diameter (SL) and stiff, large diameter (FL) was fabricated. The polymer solution was loaded into a 5 ml syringe with 0.7 diameter needle and fed at a constant rate of 0.5 ml/h using a syringe pump (Longer Pump Co., Ltd, China). Voltage of 15 kV was applied on the needle using a high-voltage power unit (Tianjing High Voltage Power Supply Co., Ltd, China). The distance between the needle tip and the collector was set at 15 cm. A rotating speed of 1400 rpm/min was used for fabricating aligned scaffolds. The prepared scaffolds were dried in vacuum overnight before further experiments.

2.5. Cell morphology visualization SEM were applied to observe the morphology of cell cultured on aligned electrospun fibrous membrane. AFSCs were cultured on different scaffolds in a-MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin for 3 d. Cells were fixed using 2.5% glutaraldehyde 2 h and then the samples were rinsed with deionized water for three times. The samples were dehydrated with graded ethanol from 10% to 100% for 15 min, and then were dried and sputter-coated with gold. The morphology of the cells was then observed using SEM at accelerating voltage of 4 kV. 2.6. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis

2.2. Characterizations of electrospun scaffolds The morphology and topography of electrospun PECUU scaffolds were analyzed using Scanning Electron Microscopy (SEM; S-4800, Hitachi Co. Ltd., Japan). The images were analyzed with NanoScope Analysis Software (Bruker) for quantification. For fiber size analysis, 200 individual fibers were measured for each scaffold. The surface wettability of scaffolds was evaluated by measuring the water contact angles with sessile drop shape method and a drop analysis system (DSA25; KRUSS, Hamburg, Germany). To measure the stiffness of the scaffolds, the fibrous membranes were cut into 15.0  3.0  0.13 mm3 specimens and evaluated through uniaxial tensile tests at a speed of 5 mm/min using an ElectropulsTM linear-torsion all-electric dynamic test instrument (E10000, Instron). The Young’s modulus of samples was calculated from the initial linear part of the stress-strain curve. The stiffness of scaffolds was also tested using a nanoindentation system (Nano Indenter G200, Agilent) using a flat indenter. At least ten indentations were conducted for each sample. 2.3. Isolation and culture of rabbit AFSCs AFSCs were isolated from AF tissue of New Zealand white rabbits (4–6 weeks old) and cultured in Alpha’s modified Eagle’s medium (a-MEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) as described previously [36]. Briefly, we harvested the rabbit spinal columns from T10 to L5 in a sterile environment and removed the surrounding muscles and ligaments carefully. Then the spinal column of each IVD was transversally sectioned. After carefully removing the NP, the pure AF tissue was minced and digested in a-MEM with Collagenase I (150 U/ml) and Collagenase II (150 U/ml) for 2–4 h. The obtained suspension was then centrifuged at 1000 rpm for 5 min, and then the cell pellet was re-suspended in a-MEM including 10% FBS, 100 U/ml penicillin, 100 lg/ml streptomycin and maintained in the humidified incubator at 37 °C with 5% CO2 at a density of 200–500 cells/ml. The culturing medium was changed every 2 d until the cells formed sub-confluence. The cells were then harvested using 0.25% trypsin-EDTA. In our research, we used the first passage of AFSCs. 2.4. Cell proliferation on PECUU scaffolds Before cell culture, the electrospun PECUU scaffolds were cut into rounded samples to fit in 96-well cell culture plates, and then were sterilized using Co-60 irradiation. Afterwards, the AFSCs were seeded on the scaffolds at a density of 5  103 cells per well. The cells were cultured in a humidified incubator at 37 °C with 5% CO2 for 1, 3, 5, 7 d. After culturing, the cells were washed with PBS, and then 20 ll MTS assay reagent (CellTiter 96 Aqueous, Promega) and 100 ll medium were added into each well. After 3 h incubation, a microplate reader (BioTek instruments, USA) was used to measure the absorbance at 490 nm.

The electrospun PECUU scaffolds were cut into circular samples fitted to the wells of a 24-well plate and sterilized using Co-60 irradiation. Then AFSCs were seeded at a density of 5  104 cells/well and incubated for 7 d. Afterwards the scaffolds were washed with PBS for three times, and the AFSCs were digested with 0.25% trypsin. The total RNA from the treated AFSCs was extracted using a TRIzol isolation system (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was performed using a Revert-Aid First-Strand cDNA Synthesis Kit (Invitrogen) and oligo (dT) primers on a reverse transcription PCR system (Eastwin Life Science). RT-qPCR was performed on a Bio-Rad CFX96 Real-Time System using the SsoFast EvaGreen Supermix Kit (Bio-Rad). The relative expression level of genes was analyzed using the 2 DDCt method normalized to the housekeeping gene GAPDH, which served as an internal control. The sequences of primers used in this study are listed in Table S1. 2.7. Immunostaining and image analysis Immunofluorescence staining was performed according to our study described previously [19]. Briefly, the cell-seeded scaffolds were washed with PBS for three times, then cells were fixed in 4% paraformaldehyde for 30 min and perforated by 0.3% Triton X-100 for 5 min. Non-specific binding was blocked by 4% bovine serum albumin (BSA) for 2 h at 37 °C. After incubation with primary antibodies (rabbit anti-YAP, Cell Signaling Technology; Mouse anti-Vinculin, Millipore Corporation) overnight, cells were incubated with the appropriate Alexa flurochrome-conjugated phalloidin. F-actin and nuclei were stained using with 647conjugated phalloidin and DAPI, respectively. Samples were embedded in ProLong Gold antifade reagent (Thermo Fisher) and visualized with confocal microscope (Zeiss LSM 880, Carl Zeiss Inc, Thornwood, NY). YAP nucleus/cytoplasm ratio was calculated by using Image-Pro Plus with the following formula:

PI

nuc =Anuc

PI

cyto =Acyto

P P where Inuc and Icyto represent the sum of the intensity values for the pixels in the nuclear and cytoplasmic region respectively, and Anuc and Acyto represent the area of the corresponding regions. Focal adhesion quantification was evaluated by ImageJ software. In brief, all the images were obtained at the same resolution and magnification according to the research described previously [37]. SUBTRACT BACKGROUND was applied to the channel corresponding to vinculin staining with a SLIDING PARABOLOID option and ROLLING BALL radius of 25 pixels. The images were enhanced by running CLAHE plug in (maximum slope = 3, histogram bins = 256, lock size = 19) followed by automatic BRIGHTNESS/CONTRAST and ENHANCE CONTRAST (saturated = 0.35). Images were finally binarized using automatic THRESHOLD

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command (default settings) and particles analysed (circularity = 0.00–0.99; size = 0.30–15). 2.8. Western blot Total protein was extracted with protein lysate (Bioytime, China) and the concentrations of total proteins were determined by BCA kit (Bioytime, China) according to manufacturer’s instructions. Proteins were electrophoresed in 10% polyacrylamide gels containing SDS, and transferred to nitrocellulose membrane from polyacrylamide gels, the nitrocellulose membranes were incubated with primary antibody (rabbit anti-Phospho-YAP, Cell Signalling Technology; mouse anti-Col-I, mouse anti-Col-II, mouse antiAggrecan, mouse anti-Actin, Abcam) overnight at 4 °C, horseradish peroxidase coupled to goat anti-rabbit IgG/anti-mouse IgG (diluted 1:1000; Abcam) for 1 h at room temperature. SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, USA) enhanced Chemiluminescent Substrate for the detection of HRP. For quantification of the expression of protein, autoradiography was performed to detect the band of the target protein (Geldoc, Bio-Rad, USA), the grayscale value of band was quantified using Image soft (Bio-Rad, USA). 2.9. Statistical analysis All data are provided as the mean ± standard deviation (SD). Statistical analysis was performed with an unpaired two-tailed Student t test for single comparisons with SPSS 18.0 (Chicago, IL, USA). For single cell analysis, a minimum of ten cells per sample was considered. One-way analysis of variance (ANOVA) was used to compare data from more than two groups. Differences were considered significantly at *p < 0.05. 3. Results 3.1. Fabrication of PECUU scaffolds with tunable stiffness and fiber sizes To mimic the structural features and gradient in fiber size of native AF tissue, aligned PECUU fibrous scaffolds with various sizes and stiffness were fabricated via electrospinning. Four types of PECUU scaffolds with controlled stiffness and fiber size: soft, small diameter (SS); stiff, small diameter (FS); soft, large diameter (SL) and stiff, large diameter (FL) was fabricated. The surface topography of aligned fibrous scaffolds was examined using SEM. The diameters of SS, FS, SL and FL were measured to be 0.54 ± 0.07, 0.52 ± 0.09, 2.76 ± 0.26 and 2.78 ± 0.32 lm, respectively (Fig. 1). The stiffness of SS, FS, SL and FL scaffold membranes were measured to be 3.5 ± 0.5, 13.5 ± 0.4, 3.9 ± 0.6 and 13.9 ± 0.6 MPa, respectively. Here, no significant difference was noted between fiber size conditions, confirming that topography could be tuned independently of intrinsic mechanics in the PECUUs (Table 1). The water contact angle of all scaffolds only slightly differed on average of 105° (Fig. S1). 3.2. Cell morphology and cell growth on PECUU fibrous scaffolds The fabricated scaffolds allowed stable cell adhesion to study the morphology changes of AFSCs in respond to scaffolds. Most cells were stably attached to scaffolds, and dramatic alternations of AFSCs were observed within 3 d of incubation (Fig. 2A). To investigate organization of actin, AFSCs were seeded on substrates and F-actin filaments were labeled with phalloidin. The results in Fig. 2B showed that AFSCs grown on large fiber scaffolds spread more widely and displayed spindle-like shapes with prominent

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and highly organized parallel actin fibers, especially on stiff scaffolds. In contrast, most AFSCs on scaffolds with small fibers were small and round with a fuzzy cytoskeleton, regardless of substrate stiffness. The orientation of the AFSCs cultured on these aligned fibrous scaffolds followed the direction of the fibers. The proliferation of AFSCs cultured on PECUU scaffolds was investigated by MTS assays at 1, 3, 5, and 7 d. No significant difference was found in proliferation among AFSCs cultured on different scaffolds in 5 d, while AFSCs appeared to grow much faster on all electrospun scaffolds than those on standard tissue culture polystyrene (TCPS) dishes (Fig. 3A). The cell spreading area is shown in Fig. 3B. The area of cell spreading increases as the fiber size increases. 3.3. Gene expression of AFSC on PECUU scaffolds Expression of Col-I, Col-II, Aggrecan, Adamts17, Sfrp2, Col5a1 and Col12a1 were studied to evaluate the efficacy of AF-related differentiation of AFSCs [10,13]. We first identified marked difference in the gene expression of Adamts17, Sfrp2, Col5a1 and Col12a1 of cells from the inner and outer regions of rabbit AF, confirming that these genes may be used to distinguish oAF cells from iAF cells (Fig. S2). Following, we investigated the effect of biophysical cues on the expression of Col-I, Col-II, Aggrecan, Adamts17, Sfrp2, Col5a1 and Col12a1 in AFSCs cultured on PECUU scaffolds. When scaffold stiffness was controlled, the expression level of the Col-I, Adamts17, Sfrp2, Col5a1 and Col12a1 genes in AFSC was relatively low on small fibers, while Col-II and Aggrecan genes were relatively high. The inverse was true for cells grown on large fibers. Moreover, when the fiber size of scaffold was kept constant, the expression of Col-I, Adamts17, Sfrp2, Col5a1 and Col12a1 genes in AFSCs increased with scaffold stiffness, however the expression of Col-II and Aggrecan genes showed an opposite trend (Fig. 4). 3.4. Mechanical property- and topography-induced FAs changes and YAP activation The expression of focal adhesions (FAs) was examined. FAs are the main hub for cell mechanosensing [31]. Immunofluorescent images of vinculin on large fibers clearly showed broad spreading along the cell margins leading to a broader area per cell, especially on stiff scaffolds, while cells on small fiber scaffolds showed vague and immature FAs with a diffuse distribution (Fig. 5A). Statistical results of cell adhesion are shown in Fig. 6A. The average number of FAs was higher in cells on large fibers, regardless of the stiffness of scaffolds. The distribution of FAs agrees with the altered morphology and cell spreading area per cell (Fig. 6B). To assess whether YAP was activated in response to mechanical and topographical cues, we stained AFSCs for YAP on scaffolds with various stiffness and fiber sizes and measured the percentage of YAP localized in nucleus. Immunofluorescence showed that both the stiffness and fiber size of scaffolds could promote YAP translocation (Fig. 7A-B). This suggests that stiffer and larger fiber size substrates increase vinculin assembly and activate YAP signaling in pre-differentiated AFSCs. The correlation between YAP activation and FAs was also investigated. After inhibiting YAP, AFSCs on fibers of all the four groups showed impaired FA maturation and confined cell area with no apparent cell death (Fig. 5B). Statistical results of number of FAs and cell area per cell are shown in Fig. 6A and B. These results indicated the critical role of YAP in FA formation and cell spreading. To determine whether region-dependent YAP localization exists in native AF tissue, the expression of YAP and phosphorylated YAP (pYAP) at different regions of AF was investigated. Immunohistochemistry staining results demonstrated a high degree of nuclear localization of YAP in the oAF, while cells within the iAF showed

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Fig. 1. The microstructure of scaffolds observed using SEM. (A) SEM images of electrospun PECUU fibrous scaffolds. Scale bars, 10 lm. (B) Fiber size distributions of PECUU scaffolds. SS: soft, small size fibers; FS: stiff, small size fibers; SL: soft, large size fibers; FL: stiff, large size fibers.

Scaffold

Young’s modulus1 (MPa)

Tensile strength1 (MPa)

Young’s modulus2 (MPa)

low levels of nuclear YAP and more pronounced cytosolic pYAP (Fig. S3A-C). The Western blot data agreed with the immunohistochemistry: The pYAP was higher in iAF than the oAF (Fig. S3D). Similar findings were seen in mouse IVD at 3 months (Fig. S4).

SS FS SL FL

3.5 ± 0.5 13.5 ± 0.4 3.9 ± 0.6 13.9 ± 0.6

2.5 ± 0.5 9.1 ± 1.1 2.2 ± 0.6 8.6 ± 1.2

4.2 ± 0.5 14.3 ± 0.6 4.7 ± 0.5 14.8 ± 0.4

3.5. Mechanical property- and topography-induced AFSC differentiation is associated with YAP activation

Table 1 Mechanical properties of electrospun PECUU scaffolds.

1 Mechanical properties measured by uniaxial tensile testing. Sample thickness, 0.1 mm; n = 4. 2 Young’s modulus measured by nanoindentation.

To study the connection between YAP and mechanical property- and topography-induced AFSC differentiation, the expression of Col-I, Col-II and Aggrecan in cells with/without treat-

Fig. 2. The morphology of AFSCs on scaffolds. (A) SEM images of AFSCs cultured on scaffolds. Scale bars, 50 lm. (B) Fluorescent images of cytoskeleton in AFSCs with F-actin stained by phalloidin staining on scaffolds. Scale bars, 20 lm.

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Fig. 3. Proliferation and spreading of AFSCs cultured on the fibrous scaffolds. (A) Proliferation of AFSCs by MTS assays with TCPS as control. *p < 0.05. (B) Quantification of cell spreading areas, *p < 0.05.

Fig.4. Expression of AF phenotypic marker genes in AFSCs cultured on PECUU scaffolds after 7 d. Gene expression was normalized to GAPDH expression. *p < 0.05.

ment YAP inhibitor verteporfin was investigated. The expression of Col-I was found to increased with the stiffness and fiber size of the scaffold, whereas the expression of Col-II and Aggrecan demonstrated an opposite trend (Fig. 8). This result is in accordance with PCR analysis shown in Fig. 4. Results also showed that AFSCs had significantly lower expression of Col-I after YAP inhibition on all of the substrates and relatively higher expression of Col-II and Aggrecan were seen in all four groups (Fig. 8). Successful suppression of YAP by verteporfin in AFSCs was verified by Western blot analysis (Fig. 9A). Verteporfin-treated AFSCs showed significantly lower expression of tendon/ligament-related markers (Col14a1, Msx2, Scx, Bgn, Mkx and Dcn) and higher expression of cartilagerelated marker Sox-9 than those without treatment after 7 d culture on tissue culture plates, demonstrating the determining role of YAP in the transcriptional program driving AFSC differentiation (Fig. 9B).

Fig. 5. YAP regulated FA formation in response to substrates. (A) Vinculin (red) and actin filaments (green) in AFSC seeded on microfibrous scaffolds observed by confocal laser scanning microscopy. (B) AFSCs cultured onto scaffolds, treated with YAP inhibitor verteporfin for 24 h and stained for vinculin (red) and phalloidin (green). Scale bars, 40 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Quantification of FA number (A) and cell spreading area (B) in a single cell cultured on scaffolds with or without the treatment of verteporfin. *p < 0.05.

Fig. 7. YAP nucleus translocation was dependent on stiffness and fiber size of scaffold. (A) Confocal laser scanning microscope images of immunofluorescently labeled YAP (red) and phalloidin (green) in AFSCs cultured on fibrous substrates. Scale bars, 40 lm. (B) Quantification of YAP nucleus/cytoplasm ratio in AFSCs. *p < 0.05. (C) Quantification of cell spreading area in a single cell cultured on scaffolds. *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion As a highly heterogeneous tissue, AF demonstrated significant variation in cellular phenotype, biochemical components, microstructure, and biomechanical characteristics along the radial direction. To reconstruct AF tissue with such a complex structure, precise control over the fibrous arrangement and heterogeneous nature of the collagen fibers was highly demanded [5,38]. In this work, we prepared fibrous PECUU scaffolds with different stiffness and fiber size to mimic the hierarchical microstructural features of native AF (Fig. S6). We found that increasing fiber size promoted AFSC spreading and FA assembly, especially on stiff scaffolds. Increasing stiffness or fiber size of scaffolds promoted YAP activa-

tion in AFSCs, which consequently enhance the up-regulation of tendon/ligament-related genes. Thus, we proposed a mechanism that matrix stiffness and fiber size of scaffold regulate the predifferentiated AFSC via the YAP signaling pathway. As compared to other organ/tissue, the compositional gradients of AF have an important effect on the regulation of its regeneration. The continuous increase (decrease) in Col-I (Aggrecan) content along the radially gradient from iAF to oAF allows a smooth transfer of intradiscal pressure by direct radial pressure from NP and cranial-caudal stretch from the separation of the two endplates [39,40]. The gradient microenvironments can provide bioactive cues for the guidance of cell growth and differentiation, maintaining the multitissue integrity [41,42]. A lack of these gradients

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Fig. 8. Mechanical property- and topography-mediated AFSC differentiation was YAP-dependent. (A) Detection of Col-I, Col-II and Aggrecan by Western blot after 24 h treatment with or without verteporfin. (B-D) Relative amounts of protein were determined by densitometric analysis. *p < 0.05.

Fig. 9. YAP activity drove tendon/ligament related gene expression. (A) Western blot analysis of YAP expression level in AFSCs treated with or without YAP inhibitor verteporfin. (B) Relative expression of tendon/ligament marker genes (Col14a1, Msx2, Scx, Bgn, Mkx, Dcn) and Sox-9 in AFSCs for 7 d of culture with or without verteporfin. * p < 0.05.

causes an inferior stress transfer across the interface, resulting in AF damage and a loss of their biological function. Therefore, the regeneration of AF must take into consideration the structural and mechanical characteristics of tissue over a wide range of length scales [38]. Each part of the substrates (composition, mechanical properties, topography, and geometry) plays a unique role in regulating the cell bioactivities [43–45]. However, how to finely adjust these biophysical parameters and optimally integrate them into substrates has been being challenging issue for the development of effective real ECM-mimicking grafts for biomedical applications.

Viswanathan et al. combined appropriate topographic features (surface/interface structure) with geometric ones (pore size) in the design of the scaffold to exert synergistic effect on cell functions [46]. Kai et al. found that both matrix stiffness and topography organization of cell-adhesive ligands could direct stem cell fate [15]. In our study, cells were cultured on scaffolds with controlled stiffness and fiber size. When scaffold stiffness was kept constant, the expression level of the oAF phenotypic marker genes (Col-I, Adamts17, Sfrp2, Col5a1 and Col12a1) in AFSC was relatively low on small fibers; the iAF phenotypic marker genes (ColII and Aggrecan) were relatively high. The inverse was found for

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cells grown on large fibers. Moreover, when the fiber size of scaffold was kept constant, the expression of oAF phenotypic marker genes in AFSCs increased with scaffold stiffness, while the expression of iAF phenotypic marker genes showed an opposite trend. Our results demonstrated that the stiffness and the fiber size of fibrous scaffolds could exert synergistic effect on the regulation of AFSC differentiation. Such substrate stiffness- and topographydependent changes of AFSC were similar to the gradient characteristics of native AF tissue which display a gradient of mechanical distribution and provide bioactive cues for AF regeneration. With an attempt to decipher why stiffness and fiber size of scaffolds had such a profound effects on AFSCs, the interaction between cells and scaffolds was examined. We found that FA significantly changes with increasing fiber size. FAs function like bridges between integrin-ECM connections and cytoskeleton [37,47]. Changes in these signals propagate through the FAs and thereby mediate behaviors of cells, including cell attachment, spreading, migration, differentiation, and stabilization of cell morphology [48]. The distribution of FAs is in accordance with morphological alternation of AFSCs. The FA data showed that the average number and grade of FA maturity was higher in cells on scaffolds with large fibers, especially on stiff substrates. Furthermore, vinculin was found to rearranged parallel along the axial cytoskeleton in cells on stiff scaffolds with large fiber size. This can be explained by the greater mechanical stimuli generated by geometric stiffness of scaffolds, which depend on the extensibility of fibers [49]. Cells can sense and generate internal forces for measuring the microenvironment. When cultured on scaffolds with increasing geometric stiffness which ranked from smallest to largest as SS, FS, SL and FL, cells sense the matrix through integrinmediated FAs, and increasing mechanical forces are generated by actin polymerization and myosin II-dependent contractility to deform the substrate [50–52]. The strong mechanical feedback from large scaffolds leads to the unfolding of talins that expose binding domains for vinculins. This further activates the RHO pathway and YAP nucleus translocation [53–55]. Interesting, although in this study we treat stiffness and fiber size of scaffolds as independent parameters, they both factor into extensibility. Due to the essential role of actomyosin cytoskeleton in mechanotransduction, we also studied the pathway in which mechanical signals were transduced into biological outcomes and the ultimate impacts of these signals on gene expression. The transcriptional coactivators YAP and TAZ were recently recognized as key mediators of the biological effects observed in response to ECM elasticity and cell shape [56–58]. Recent studies demonstrated the critical role of YAP signaling in regulation of mechanosensitive MSC differentiation [59–61]. Shamik et al. reported the necessity of YAP activation in topography-mediated transition of model endothelium towards a highly proliferative and migratory phenotype [62]. Bao et al. demonstrated that cellular volume and matrix stiffness direct stem cell behavior in 3D microniche through YAP/TAZ signaling [63]. These studies highlighted the mechanotransductive mechanisms acting upstream of YAP transcriptional activity via Rho GTPases, cytoskeletal contractility, and F-actin mechanics. Our study showed that large-sized and high-stiffness substrates enhanced the amount of vinculin assembly in pre-differentiated AFSCs and activate YAP signaling. We identified a new mechanism that involves YAP in the regulation of AFSC activities via stiffness and fiber size of substrates. This differentiation-associated YAP nuclear translocation was reported in many other types of stem cells and could be likely due to changes in composition of structural ECM proteins. To determine whether YAP localization is similarly regulated by the anatomical features of AF tissue, we investigated the regional expression of YAP and pYAP of AF. Clearly, YAP is mostly located in the nuclear in cells within oAF consisting of relatively large size collagen fibrils and stiff ECM, while cells in iAF had high

levels of phosphorylated and cytosolic YAP. Similar patterns were observed during mouse embryonic limb development, where YAP was mostly in nucleus in cells in the perichondrium composing of large collagen fibrils and stiff ECM. Cells in cartilage (relatively small collagen fibrils and soft ECM) showed the exact opposite behavior [34,64]. These findings agree with our in vitro observations that YAP is regulated in a mechanical and topographical fashion. Integrin-FA signaling was reported to control Hippo pathway by phosphorylating large tumor suppressor (LATS) kinases via Src [65]. Recent evidence suggested possible interplays between YAP/TAZ and integrin-FA signaling. We here investigated the connection between YAP activation and the presence of FAs. Interestingly, AFSCs on fibers of all the four groups had impaired FA maturation after YAP inhibition. This agrees with several studies showing that YAP was necessary to promote FA formation [30,66]. Cells lacking YAP showed complete disappearance of typical FA spikes, implying the inability of YAP-inhibited cells to interact with the surrounding ECM [28,31]. Here, we showed that YAP is involved in a regulatory mechanism in which scaffolds with high stiffness and large fiber size direct AFSC toward oAF cell lineage by increasing the YAP nuclear translocation. For investigation of YAP’s specific contribution towards AFSC differentiation, we analyzed the protein expression of Col-I, Col-II, and Aggrecan after treatment with or without YAP inhibitor. The results showed that AFSCs on all of the substrates had significantly lowered expression of Col-I after YAP inhibition, while expression of Col-II and Aggrecan was relatively high in all four groups after YAP inhibition. Furthermore, YAP is a transcriptional factor that shuttles between the cytoplasm and the nucleus that associate with several promoter-specific transcription factors. Recent studies have shown that mechanical signals stimulate YAP activation of periodontal ligament cells and induce them to differentiate into myofibroblasts to initiate periodontal tissue remodeling [35]. Our results here discovered for the first time that the activation of YAP could promote AFSC to differentiate into tendon/ligament-like cells with high expression of Col-I which dominate in cells in oAF. The results indicate that engineered AF scaffolds should ideally possess a gradient both in stiffness and fiber size along the radical direction to stimulate AFSC to differentiate into different region-specific AF cells and to secrete corresponding ECM to promote AF tissue regeneration. Structural recapitulation of AF tissue remains a major challenge for AF tissue engineering. Naturally derived materials such as decellularized ECM are generally high in bioactivity, but lack controllable structures and mechanical properties. On the contrary, synthetic materials often have low bioactivity, but their structures and mechanical properties can be easily manipulated. Therefore, a combination of natural and synthetic materials may allow the development of scaffolds with robust mechanical properties and good bioactivity for AF regeneration in the future. Despite of these obstacles in clinical implementation, we expect our findings to have broad implications for engineering multilayered tissues because FAs and YAP are present in many types of tissue and are believed to be significantly affected by the biophysical cues of substrates.

5. Conclusions Fibrous scaffolds with tunable stiffness and fiber size were fabricated using electrospinning to mimic the anatomy characteristics of native AF tissue. The engineered scaffolds enabled the discovery that scaffolds with large fiber size promoted the maturation of FAs and spreading of cultured AFSCs, especially on stiff scaffolds. When scaffold stiffness was fixed, the expression level of the oAF pheno-

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typic marker genes in AFSC was relatively low on small fibers, while iAF phenotypic genes were relatively high. The opposite trend was observed for cells grown on large fibers. Moreover, when the fiber size of scaffold was kept constant, the expression of oAF phenotypic marker genes in AFSCs increased with scaffold stiffness, however the expression of iAF phenotypic genes showed an opposite trend. As a consequence, stiffness and the fiber size of scaffolds are both vital cues of stem cell microenvironment. Consistent with this mechanistic hypothesis, chemical disruption of YAP signaling blocked FA assembly and the tendency of AFSC to differentiate into various types of AF-like cells. The present study affords an in-depth comprehension of materiobiology, and will be helpful for explain the mechanism of YAP mechanosensing in AF in response to biophysical effects of materials.

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Acknowledgments [19]

This work was supported by the National Key R&D Program of China (2016YFC1100203), National Natural Science Foundation of China (81672213, 31530024, 81702200 and 31700854), Jiangsu Provincial Special Program of Medical Science (BL2012004), Jiangsu Provincial Clinical Orthopedic Center, Key Laboratory of Stem Cells and Biomedical Materials of Jiangsu Province and Chinese Ministry of Science and Technology, the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions and Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18_2529). Conflict of interests

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The authors declare no conflict of interest. [25]

Appendix A. Supplementary data [26]

Supplementary data to this article can be found online at https://doi.org/10.1016/j.actbio.2019.05.013.

[27]

References

[28]

[1] G. Fontana, E. See, A. Pandit, Current trends in biologics delivery to restore intervertebral disc anabolism, Adv. Drug Deliv. Rev. 84 (2015) 146–158. [2] P. Finch, Technology insight: imaging of low back pain, Nat. Clin. Pract. Rheumatol. 2 (10) (2006) 554–561. [3] N.L. Nerurkar, B.M. Baker, S. Sen, E.E. Wible, D.M. Elliott, R.L. Mauck, Nanofibrous biologic laminates replicate the form and function of the annulus fibrosus, Nat. Mater. 8 (12) (2009) 986–992. [4] J.C. Yang, X.L. Yang, L. Wang, W. Zhang, W.B. Yu, N.X. Wang, B.A. Peng, W.F. Zheng, G. Yang, X.Y. Jiang, Biomimetic nanofibers can construct effective tissue-engineered intervertebral discs for therapeutic implantation, Nanoscale 9 (35) (2017) 13095–13103. [5] G.J. Rosenberg, A.J.M. Yee, W.M. Erwin, Bedside to bench and back to bedside: translational implications of targeted intervertebral disc therapeutics, J. Orthopaedic Transl. 10 (2017) 18–27. [6] F.J. Lyu, K.M. Cheung, Z. Zheng, H. Wang, D. Sakai, V.Y. Leung, IVD progenitor cells: a new horizon for understanding disc homeostasis and repair, Nat. Rev. Rheumatol. 15 (2) (2019) 102–112. [7] R. Nakamichi, Y. Ito, M. Inui, N. Onizuka, T. Kayama, K. Kataoka, H. Suzuki, M. Mori, M. Inagawa, S. Ichinose, M.K. Lotz, D. Sakai, K. Masuda, T. Ozaki, H. Asahara, Mohawk promotes the maintenance and regeneration of the outer annulus fibrosus of intervertebral discs, Nat. Commun. 7 (2016) 12503. [8] G. Pattappa, Z. Li, M. Peroglio, N. Wismer, M. Alini, S. Grad, Diversity of intervertebral disc cells: phenotype and function, J. Anat. 221 (6) (2012) 480– 496. [9] C.K. Kepler, R.K. Ponnappan, C.A. Tannoury, M.V. Risbud, D.G. Anderson, The molecular basis of intervertebral disc degeneration, Spine J. 13 (3) (2013) 318– 330. [10] J. Li, C. Liu, Q. Guo, H. Yang, B. Li, Regional variations in the cellular, biochemical, and biomechanical characteristics of rabbit annulus fibrosus, PLoS ONE 9 (3) (2014) e91799. [11] S. Ghazanfari, A. Khademhosseini, T.H. Smit, Mechanisms of lamellar collagen formation in connective tissues, Biomaterials 97 (2016) 74–84. [12] G.G. van den Akker, D.A. Surtel, A. Cremers, S.M. Richardson, J.A. Hoyland, L.W. van Rhijn, J.W. Voncken, T.J. Welting, Novel immortal cell lines support cellular

[29]

[30] [31]

[32]

[33]

[34]

[35]

[36] [37]

[38]

[39]

263

heterogeneity in the human annulus fibrosus, PLoS ONE 11 (1) (2016) e0144497. G.G.H. van den Akker, M.I. Koenders, F.A.J. van de Loo, P. van Lent, E. Blaney Davidson, P.M. van der Kraan, Transcriptional profiling distinguishes inner and outer annulus fibrosus from nucleus pulposus in the bovine intervertebral disc, Eur. Spine J. 26 (8) (2017) 2053–2062. A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, Matrix elasticity directs stem cell lineage specification, Cell 126 (4) (2006) 677–689. K. Ye, X. Wang, L. Cao, S. Li, Z. Li, L. Yu, J. Ding, Matrix stiffness and nanoscale spatial organization of cell-adhesive ligands direct stem cell fate, Nano Lett. 15 (7) (2015) 4720–4729. H. Yuan, Y. Zhou, M.S. Lee, Y. Zhang, W.J. Li, A newly identified mechanism involved in regulation of human mesenchymal stem cells by fibrous substrate stiffness, Acta Biomater. 42 (2016) 247–257. M. Bhattacharjee, S. Miot, A. Gorecka, K. Singha, M. Loparic, S. Dickinson, A. Das, N.S. Bhavesh, A.R. Ray, I. Martin, S. Ghosh, Oriented lamellar silk fibrous scaffolds to drive cartilage matrix orientation: towards annulus fibrosus tissue engineering, Acta Biomater. 8 (9) (2012) 3313–3325. B.K. Bhunia, D.L. Kaplan, B.B. Mandal, Silk-based multilayered angle-ply annulus fibrosus construct to recapitulate form and function of the intervertebral disc, Proc. Natl. Acad. Sci. U.S.A. 115 (3) (2018) 477–482. C. Liu, C. Zhu, J. Li, P. Zhou, M. Chen, H. Yang, B. Li, The effect of the fibre orientation of electrospun scaffolds on the matrix production of rabbit annulus fibrosus-derived stem cells, Bone Res. 3 (2015) 15012. Q.P. Guo, C. Liu, J. Li, C.H. Zhu, H.L. Yang, B. Li, Gene expression modulation in TGF-3-mediated rabbit bone marrow stem cells using electrospun scaffolds of various stiffness, J. Cell Mol. Med. 19 (7) (2015) 1582–1592. C. Zhu, J. Li, C. Liu, P. Zhou, H. Yang, B. Li, Modulation of the gene expression of annulus fibrosus-derived stem cells using poly(ether carbonate urethane)urea scaffolds of tunable elasticity, Acta Biomater. 29 (2016) 228–238. H. Jeon, S. Koo, W.M. Reese, P. Loskill, C.P. Grigoropoulos, K.E. Healy, Directing cell migration and organization via nanocrater-patterned cell-repellent interfaces, Nat. Mater. 14 (9) (2015) 918–923. M.J. Dalby, N. Gadegaard, R. Tare, A. Andar, M.O. Riehle, P. Herzyk, C.D.W. Wilkinson, R.O.C. Oreffo, The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder, Nat. Mater. 6 (12) (2007) 997–1003. J. Du, X.F. Chen, X.D. Liang, G.Y. Zhang, J. Xu, L.R. He, Q.Y. Zhan, X.Q. Feng, S. Chien, C. Yang, Integrin activation and internalization on soft ECM as a mechanism of induction of stem cell differentiation by ECM elasticity, Proc. Natl. Acad. Sci. U.S.A. 108 (23) (2011) 9466–9471. S. Khetan, M. Guvendiren, W.R. Legant, D.M. Cohen, C.S. Chen, J.A. Burdick, Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels, Nat. Mater. 12 (5) (2013) 458–465. M. Elsaadany, K. Winters, S. Adams, A. Stasuk, H. Ayan, E. Yildirim-Ayan, Equiaxial Strain modulates adipose-derived stem cell differentiation within 3D biphasic scaffolds towards annulus fibrosus, Sci. Rep. 7 (1) (2017) 12868. Y.L. Li, Y. Xiao, C.S. Liu, The horizon of materiobiology: a perspective on material-guided cell behaviors and tissue engineering, Chem. Rev. 117 (5) (2017) 4376–4421. T. Panciera, L. Azzolin, M. Cordenonsi, S. Piccolo, Mechanobiology of YAP and TAZ in physiology and disease, Nat. Rev. Mol. Cell Biol. 18 (12) (2017) 758– 770. B. Zhao, K. Tumaneng, K.L. Guan, The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal, Nat. Cell Biol. 13 (8) (2011) 877–883. A. Totaro, T. Panciera, S. Piccolo, YAP/TAZ upstream signals and downstream responses, Nat. Cell Biol. 20 (8) (2018) 888–899. G. Halder, S. Dupont, S. Piccolo, Transduction of mechanical and cytoskeletal cues by YAP and TAZ, Nat. Rev. Mol. Cell Biol. 13 (9) (2012) 591–600. S. Dupont, L. Morsut, M. Aragona, E. Enzo, S. Giulitti, M. Cordenonsi, F. Zanconato, J. Le Digabel, M. Forcato, S. Bicciato, N. Elvassore, S. Piccolo, Role of YAP/TAZ in mechanotransduction, Nature 474 (7350) (2011) 179–183. J. Dong, G. Feldmann, J. Huang, S. Wu, N. Zhang, S.A. Comerford, M.F. Gayyed, R. A. Anders, A. Maitra, D. Pan, Elucidation of a universal size-control mechanism in Drosophila and mammals, Cell 130 (6) (2007) 1120–1133. A. Karystinou, A.J. Roelofs, A. Neve, F.P. Cantatore, H. Wackerhage, C. De Bari, Yes-associated protein (YAP) is a negative regulator of chondrogenesis in mesenchymal stem cells, Arthritis Res. Ther. 17 (2015). Y. He, H. Xu, Z. Xiang, H. Yu, L. Xu, Y. Guo, Y. Tian, R. Shu, X. Yang, C. Xue, M. Zhao, Y. He, X. Han, D. Bai, YAP regulates periodontal ligament cell differentiation into myofibroblast interacted with RhoA/ROCK pathway, J. Cell. Physiol. 234 (4) (2019) 5086–5096. C. Liu, Q. Guo, J. Li, S. Wang, Y. Wang, B. Li, H. Yang, Identification of rabbit annulus fibrosus-derived stem cells, PLoS ONE 9 (9) (2014) e108239. G. Nardone, J. Oliver-De La Cruz, J. Vrbsky, C. Martini, J. Pribyl, P. Skladal, M. Pesl, G. Caluori, S. Pagliari, F. Martino, Z. Maceckova, M. Hajduch, A. SanzGarcia, N.M. Pugno, G.B. Stokin, G. Forte, YAP regulates cell mechanics by controlling focal adhesion assembly, Nat. Commun. 8 (2017) 15321. M. Peroglio, L.S. Douma, T.S. Caprez, M. Janki, L.M. Benneker, M. Alini, S. Grad, Intervertebral disc response to stem cell treatment is conditioned by disc state and cell carrier: an ex vivo study, J. Orthopaedic Transl. 9 (2017) 43–51. G. Chu, C. Shi, J. Lin, S. Wang, H. Wang, T. Liu, H. Yang, B. Li, Biomechanics in annulus fibrosus degeneration and regeneration, Adv. Exp. Med. Biol. 1078 (2018) 409–420.

264

G. Chu et al. / Acta Biomaterialia 92 (2019) 254–264

[40] C.C. Guterl, E.Y. See, S.B. Blanquer, A. Pandit, S.J. Ferguson, L.M. Benneker, D.W. Grijpma, D. Sakai, D. Eglin, M. Alini, J.C. Iatridis, S. Grad, Challenges and strategies in the repair of ruptured annulus fibrosus, Eur. Cells Mater. 25 (2013) 1–21. [41] K.L. Moffat, W.H.S. Sun, P.E. Pena, N.O. Chahine, S.B. Doty, G.A. Ateshian, C.T. Hung, H.H. Lu, Characterization of the structure-function relationship at the ligament-to-bone interface, Proc. Natl. Acad. Sci. U.S.A. 105 (23) (2008) 7947– 7952. [42] Q. Hu, M. Liu, G. Chen, Z. Xu, Y. Lv, Demineralized bone scaffolds with tunable matrix stiffness for efficient bone integration, ACS Appl. Mater. Interfaces 10 (33) (2018) 27669–27680. [43] E. Bier, E.M. De Robertis, Embryo development. BMP gradients: a paradigm for morphogen-mediated developmental patterning, Science 348 (6242) (2015). aaa5838. [44] P.C. Dave, P. Dingal, D.E. Discher, Combining insoluble and soluble factors to steer stem cell fate, Nat. Mater. 13 (6) (2014) 532–537. [45] Q.B. Lv, X. Gao, X.X. Pan, H.M. Jin, X.T. Lou, S.M. Li, Y.Z. Yan, C.C. Wu, Y. Lin, W.F. Ni, X.Y. Wang, A.M. Wu, Biomechanical properties of novel transpedicular transdiscal screw fixation with interbody arthrodesis technique in lumbar spine: a finite element study, J. Orthopaedic Transl. 15 (2018) 50–58. [46] P. Viswanathan, M.G. Ondeck, S. Chirasatitsin, K. Ngamkham, G.C. Reilly, A.J. Engler, G. Battaglia, 3D surface topology guides stem cell adhesion and differentiation, Biomaterials 52 (2015) 140–147. [47] L. Lorenz, J. Axnick, T. Buschmann, C. Henning, S. Urner, S. Fang, H. Nurmi, N. Eichhorst, R. Holtmeier, K. Bodis, J.H. Hwang, K. Mussig, D. Eberhard, J. Stypmann, O. Kuss, M. Roden, K. Alitalo, D. Haussinger, E. Lammert, Mechanosensing by beta1 integrin induces angiocrine signals for liver growth and survival, Nature 562 (7725) (2018) 128–132. [48] A. Carisey, C. Ballestrem, Vinculin, an adapter protein in control of cell adhesion signalling, Eur. J. Cell Biol. 90 (2–3) (2011) 157–163. [49] M.E. Piroli, E. Jabbarzadeh, Matrix stiffness modulates mesenchymal stem cell sensitivity to geometric asymmetry signals, Ann. Biomed. Eng. 46 (6) (2018) 888–898. [50] J.S. Mo, F.X. Yu, R. Gong, J.H. Brown, K.L. Guan, Regulation of the Hippo-YAP pathway by protease-activated receptors (PARs), Genes Dev. 26 (19) (2012) 2138–2143. [51] K.T. Furukawa, K. Yamashita, N. Sakurai, S. Ohno, The epithelial circumferential actin belt regulates YAP/TAZ through nucleocytoplasmic shuttling of merlin, Cell Rep. 20 (6) (2017) 1435–1447. [52] D. Lachowski, E. Cortes, D. Pink, A. Chronopoulos, S.A. Karim, Jennifer P. Morton, A.E. Del Rio Hernandez, Substrate rigidity controls activation and durotaxis in pancreatic stellate cells, Scientific Rep. 7 (1) (2017) 2506. [53] T. Zhang, T. Gong, J. Xie, S.Y. Lin, Y. Liu, T.F. Zhou, Y.F. Lin, Softening substrates promote chondrocytes phenotype via RhoA/ROCK pathway, ACS Appl. Mater. Interfaces 8 (35) (2016) 22884–22891.

[54] W.J. Hadden, J.L. Young, A.W. Holle, M.L. McFetridge, D.Y. Kim, P. Wijesinghe, H. Taylor-Weiner, J.H. Wen, A.R. Lee, K. Bieback, B.N. Vo, D.D. Sampson, B.F. Kennedy, J.P. Spatz, A.J. Engler, Y.S. Choi, Stem cell migration and mechanotransduction on linear stiffness gradient hydrogels, Proc. Natl. Acad. Sci. U.S.A. 114 (22) (2017) 5647–5652. [55] A. del Rio, R. Perez-Jimenez, R. Liu, P. Roca-Cusachs, J.M. Fernandez, M.P. Sheetz, Stretching single talin rod molecules activates vinculin binding, Science 323 (5914) (2009) 638–641. [56] Z. Meng, Y. Qiu, K.C. Lin, A. Kumar, J.K. Placone, C. Fang, K.C. Wang, S. Lu, M. Pan, A.W. Hong, T. Moroishi, M. Luo, S.W. Plouffe, Y. Diao, Z. Ye, H.W. Park, X. Wang, F.X. Yu, S. Chien, C.Y. Wang, B. Ren, A.J. Engler, K.L. Guan, RAP2 mediates mechanoresponses of the Hippo pathway, Nature 560 (7720) (2018) 655–660. [57] K.C. Lin, H.W. Park, K.L. Guan, Deregulation and therapeutic potential of the hippo pathway in cancer, Annu. Rev. Cancer Biol. 2 (2018) 59–79. [58] J.H. Koo, K.L. Guan, Interplay between YAP/TAZ and metabolism, Cell Metab. 28 (2) (2018) 196–206. [59] A. Elosegui-Artola, R. Oria, Y.F. Chen, A. Kosmalska, C. Perez-Gonzalez, N. Castro, C. Zhu, X. Trepat, P. Roca-Cusachs, Mechanical regulation of a molecular clutch defines force transmission and transduction in response to matrix rigidity, Nat. Cell Biol. 18 (5) (2016) 540–548. [60] J.Y. Shiu, L. Aires, Z. Lin, V. Vogel, Nanopillar force measurements reveal actincap-mediated YAP mechanotransduction, Nat. Cell Biol. 20 (3) (2018) 262– 271. [61] J.K. Hu, W. Du, S.J. Shelton, M.C. Oldham, C.M. DiPersio, O.D. Klein, An FAKYAP-mTOR signaling axis regulates stem cell-based tissue renewal in mice, Cell Stem Cell 21 (1) (2017) 91–106. [62] S. Mascharak, P.L. Benitez, A.C. Proctor, C.M. Madl, K.H. Hu, R.E. Dewi, M.J. Butte, S.C. Heilshorn, YAP-dependent mechanotransduction is required for proliferation and migration on native-like substrate topography, Biomaterials 115 (2017) 155–166. [63] M. Bao, J. Xie, N. Katoele, X. Hu, B. Wang, A. Piruska, W.T.S. Huck, Cellular volume and matrix stiffness direct stem cell behavior in a 3D microniche, ACS Appl. Mater. Interfaces (2018). [64] T. Liang, L.L. Zhang, W. Xia, H.L. Yang, Z.P. Luo, Individual collagen fibril thickening and stiffening of annulus fibrosus in degenerative intervertebral disc, Spine 42 (19) (2017) E1104–E1111. [65] H. Nakajima, K. Yamamoto, S. Agarwala, K. Terai, H. Fukui, S. Fukuhara, K. Ando, T. Miyazaki, Y. Yokota, E. Schmelzer, H.G. Belting, M. Affolter, V. Lecaudey, N. Mochizuki, Flow-dependent endothelial YAP regulation contributes to vessel maintenance, Dev. Cell 40 (6) (2017). 523-536 e6. [66] B.M. Baker, B. Trappmann, W.Y. Wang, M.S. Sakar, I.L. Kim, V.B. Shenoy, J.A. Burdick, C.S. Chen, Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments, Nat. Mater. 14 (12) (2015) 1262–1268.