Controlling the Multiscale Network Structure of Fibers To Stimulate

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Surfaces, Interfaces, and Applications

Controlling Multiscale Network Structure of Fibers to Stimulate Wound Matrix Rebuilding by Fibroblast Differentiation Yi Li, Zecong Xiao, Yajiao Zhou, Shen Zheng, Ying An, Wen Huang, Huacheng He, Yao Yang, Shengyu Li, Yanxin Chen, Jian Xiao, and Jiang Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b06439 • Publication Date (Web): 28 Jun 2019 Downloaded from pubs.acs.org on July 19, 2019

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ACS Applied Materials & Interfaces

Controlling Multiscale Network Structure of Fibers to Stimulate Wound Matrix Rebuilding by Fibroblast Differentiation

Yi Li1,3#, Zecong Xiao1#, Yajiao Zhou1, Shen Zheng1, Ying An1, Wen Huang1, Huacheng He2*, Yao Yang2, Shengyu Li2, Yanxin Chen2, Jian Xiao1*, Jiang Wu1*

1School

of Pharmaceutical Sciences

Wenzhou Medical University, Wenzhou, Zhejiang 325035, P.R. China

2College

of Chemistry and Materials Engineering

Wenzhou University, Wenzhou, Zhejiang 325027, P.R. China

3The

Third Affiliated Hospital of Wenzhou Medical University

Wenzhou, Zhejiang 325200, P.R. China

*Corresponding Author: JW ([email protected]); XX ([email protected]); HH ([email protected]);

# The authors contribute equally to this work

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ABSTRACT Extracellular matrix (ECM) play a double-edged sword role for controlling the differentiation of fibroblast toward contractile myofibroblast in the wound healing process. However, the exact structure-function relationship between ECM morphology and fibroblast behaviors still remains unclear. To better understand this relationship, herein we designed and prepared a series of biocompatible polycaprolactone (PCL)-based fibers with different fiber diameters (nano vs. micro) and different alignments (random vs. aligned) using a simple electrospinning, with a particular attention to the morphological effect of PCL fiber scaffolds on guiding fibroblast behaviors. Microfibers with the larger fiber diameters induce less cell spreading, adhesion, differentiation, and migration due to its lower surface tension. In contrast, nanofibers will retain fibroblast cells with typical spindle-shapes and promote the expression of focal adhesive proteins through the integrin pathway. Furthermore, nanofibers up-regulate the expression α-smooth muscle actin (α-SMA), transforming growth factor (TGF-β), and vimentin filaments, confirming that the size change of PCL fiber matrix from micrometers to nanometers indeed alter fibroblast differentiation to activate more α-SMA-expressed contractile myofibroblast. Such fiber sizedependent fibroblast behavior is largely attributed to the enhanced surface tension from the dressing matrix, which helps to promote the conversion of fibroblasts to myofibroblasts via either tissue regeneration or fibrosis. Therefore, this work further indicated the reason of re-arrangement of collagen from nano tropocollagen to micro collagen bundles during the wound healing process can reverses fibroblasts to myofibroblasts from motivated to demise. This finding allows us to achieve the structural-based design of new fibrous matrix for promoting wound healing and tissue regeneration. Keywords: wound healing, fibroblast differentiation, fibrosis, nanofiber, microfiber, collagen deposition



1 Introduction Fibroblast play important physiological or pathological roles in both wound regeneration1 and fibrosis formation2. Fibroblast are non-epithelial, non-endothelial, non-parenchymal nor mesothelial cells, typically of mesenchymal origin3. However, they could originate from epithelial4, endothelial-to-mesenchymal transition5 as well as mesothelial6. Fibroblast sensitively respond to tissue microenvironments and then rebuild their own extracellular matrix (ECM) components by “Fibroblast-to-Myofibroblast” differentiation7, that is primarily induced by transforming growth factor β1 released by both paracrine release of epithelial, inflammatory, or tumor cells and autocrine secretion. Mechanical factors can also modulate the fibroblast differentiation by providing tensional forces on the fibroblast cytoskeleton. Upon early wound region, the fibroblasts migrating to the wound matrix, where they proliferate and regenerate new collagen deposition. This gradually increased stiffness of extracellular matrix (ECM) force the “Fibroblast-to-Myofibroblast” transition which actively close the wound8. Once epithelium would have covered the whole wound, myofibroblasts disappeared by apoptosis along with wound healed. However, under pathological conditions, myofibroblast refuses to undergo apoptosis in time but overproduce ECM forming fibrotic disease such as fibrosis9. The reversal of myofibroblast from motivated to demise along with wound healing process has not been documented and massive apoptosis of myofibroblast occurring after re-epithelization upon wound regeneration leading to myofibroblast group suicide still remain unclear10-11. Despite the extensive studies on the stiffness changes, with less success of explaining the myofibroblast apoptosis, we assume that the nano to micro morphology changes of the ECM upon the wound healing is the dark force to the reversal of myofibroblast12. As the remolding of the wound matrix, production of collagen13 plays an essential role in bridging the wound gap as well as improving the wound strength. It changes from single collagen fibrils ranging from 50-200nm nanoscale to tightly cross-linked micro-fibrils collagen bundles >1μm14. In the end of wound regeneration, thick strong crosslinked microscale collagen bundles become regularly arranged and perpendicular on the plane of wound15-16. During the process, considering the ECM microenvironment plays crucial role in modulation of cellular morphology and migration17-20, we therefore hypothesize that this nano-to-micro matrix changes may affect both initiation and termination of myofibroblast progression21-22.


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To address this hypothesis, fibroblast (NIH 3T3) were seeded on electrospinning PCL (polycaprolactone, FDA approved biomedical aliphatic polyesters) fibrous matrices with nanopatterned (300 nm) random fibers and micro-patterned (1-2 μm) random fibers together with aligned oriented nanofibers. These fibroblasts can build their own structures by using PCL fibers as a 3D template over long-term culture. Our results showed that on nanofibers, fibroblast differentiated to myofibroblast cells by forming a spread, stellate morphology with multiple attachment bonding sites between the cells and the nanofibers. While with the increase of diameters of fibers to micro range, the cells become less spread. Also, the decreased expression of α-SMA further confirmed the impediment of fibroblast to myofibroblast transformation from nano to micro structure. This finding for the first time using the electrospinning method to modulate the ECM remolding through the regeneration process, enable a deeper understanding of wound fibroblast differentiation and the potential to serve as instructive matrix to facilitate wound repair and regeneration.

2 Experimental section 2.1 Electrospun PCL fibers of different diameters Different diameters of PCL fibers were fabricated by using an electrospinning apparatus. In order to obtain a 6% w/w PCL polymer solution, 0.68 mg polycaprolactone solute (PCL, Mw=80kDa, Sigma) was dissolved in 7 ml of HFIP and well mixed; Then the solution was loaded into a 10 mL syringe (BD) and the syringe pump was attached to a high voltage statitron. The typical parameters of fabricated electrospinning fibers are listed as follows. Briefly, the suitable electric voltage was 12 kV, the distance between the injector nozzle and the collector was fixed at 15 cm and the electrospinning environment was at room temperature. These parameters were applicable for all electrospinning samples. Dissimilarly, by varying the feed rate to obtain different diameters of random PCL fibers and mimic the cell microenvironment. The nanofiber was fabricated at a rate of 2.88 mm/h, and the feed rate of microfiber was about 20 times faster than nanofiber. The various diameters of random structured fibers were collected by using a planar collector, and the aligned fiber was collected by using a rotating cylindrical collector at 1600 r/min. 2.2 Physical Characterization of fibers A scanning electron microscopy (SEM, VEGA3 TESCAN) were applied to characterize the morphology of different fibrous scaffold. Prior to imaging, the fibrous specimens were dried with nitrogen and sputtered with gold for 60 s with Desk II cold sputter coater (Denton Vacuum,



Morristown, NJ). For each sample, no less than three images were analyzed, and the average fiber diameter was manually measured and calculated by using Image-pro plus. Finally, the results are manifested as mean ± standard deviation. 2.3 Cell culture and seeding NIH 3T3 mouse fibroblast cells (ATCC, Manassas, VA, USA) were maintained in growth media-dulbecco’s modified eagle medium (DMEM, Gibco) supplemented with 10%fetal bovine serum (FBS, Gibco) and 100 U/mL penicillin-streptomycin solution (PS, Gibco). Then the NIH 3T3 cells were cultured at 37 oC under 5% CO2/95% air humidified incubator. Before plating on the fibers, each group PCL fiber was sterilized in UV for 45 min and soaked in media 30 min. Afterwards, sub-confluent cells were washed three times by PBS and trypsinized by 0.25% trypsin and plated on micro-fiber, nano-fiber, aligned-fiber and TCP at a density of 5×104 cells per cm2. 2.4 Cell viability Live cell staining of fibroblast cells on microfiber, nanofiber, Aligned-fiber and TCPs using the fluorescein diacetate (FDA, Sigma-Aldrich), a fluorescence staining dye of living cells. After 24 h and 72 h, the DMEM complete medium was removed and the specimens were rinsed gently with PBS (3×). Then, 1 mL of FDA (4 μg/ ml) was added per well and incubated for 30 min at 37 ℃. After it, the samples were rinsed three times with PBS and the number of viable cells were observed using a fluorescence microscope (A1 PLUS, Tokyo, Japan, Nikon) at excitation wavelengths of 488 nm (green). 2.5 Cellular morphology In order to examine the morphology of the cell adhering on the surface of different PCL fibers, the cell was rinsed by PBS (3×) and trypsinized and re-suspended in 1 mL DMEM. Subsequently, PCL fibers were stuck to the TCP and placed in a 24-well culture plate supplemented with 10% FBS low glucose DMEM complete medium, then the cell solution was added to form final cell density of 5×104 per cm2. The culture condition was described early before. After incubating for one day and three days respectively, the DMEM complete medium was removed and rinsed with PBS (3×), and was fixed by glutaraldehyde solution for 4 h. At last, the specimen was lyophilized for scanning electron microscopy (SEM) observation. 2.6 Atomic force microscopy (AFM) characterization AFM imaging was used to generate 3D images of the microfiber and nanofiber scaffolds. And


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the density of fibrous scaffolds was quantified from the AFM images. The local area of fibrous scaffolds was captured. And the force-distance curve of adhesion force is obtained by AFM tip retract from approaching after the microfiber and nanofiber surface. Statistical analysis was collected through several random locations directly from the micro- or nanofiber surface. 2.7 Mechanical property test The mechanical test of the microfiber, nanofiber and aligned fiber were all conducted by the experimental apparatus, which was assembled with load cell of 50 N. Also, the pulling speed was controlled in 15 mm/min. Each of fibrous scaffolds was cut into rectangular specimens of 25 mm (width) × 50 mm (length). The spinning time was performed as before, and every scaffold specimen was tested at least five times at the same condition. The results of average value of maximum tensile stress and elastic modulus were then calculated. 2.8 Hydrophilicity measurement The hydrophilicity of surface morphology of nanofiber, microfiber and aligned fiber were tested by the contact angle apparatus (KRUSS, DSA30). Briefly, a drop of water was dropped onto the different fiber (18 cm × 18 cm) films. After contacting with each other, the image of water drop was captured with the same time scale, and the water contact angle and contact distance were calculated. 2.9 qRT-PCR analysis Genes involved in fibroblast and myofibroblast were selected to view the surface topography of micro- and nanofibers regarding the scaffold of fibroblast differentiation. The major maker for myofibroblast are: α-smooth muscle actin (α-SMA); focal adhesion genes: Integrin α1, paxillin and focal adhesion kinase (FAK); extracellular protein genes: fibronectin (FN). All of these genes are related to the fibroblast differentiation. Cells were seeded on the microfiber, nanofiber, aligned fiber and TCP for one day and three days respectively, the total RNA was extracted from these samples by using Tripure lsolation Reagent (Roche, US) according to the manufacturer’s protocol. In addition, the total RNA was confirmed at 260 nm and 280 nm by using the Nanodrop 2000 (Thermo Scientific). cDNA synthesis was performed by using RNA PCR kit, and qPCR amplification was performed by using SYBR green (Roche) detection. The method used was 40 cycles of PCR, after the initial denaturation step of 2 min at 95 oC, the subsequent step was 95 oC for 15 s and 60 oC for 30 s. Finally, relative quantification of results was conducted by applying the standard 2−∆∆Ct method



and GAPDH which is used for normalization. 2.10 Immunofluorescence staining NIH 3T3 cells were grown on different fibrous scaffolds for 3 days, then fibers were rinsed with PBS (3×) and fixed in 4% formaldehyde solution. After 15 minutes, the samples were blocked with the use of 5% bovine serum albumin (BSA) (Beyotime) that contains 0.5% Triton X-100 (Amresco) for 30 minutes under room temperature. Primary antibodies anti-vimentin (ab92547, 1:300, abcam), anti-collagen Ⅲ (ab7778, 1:300, abcam), α-SMA (ab7817, 1:200, abcam), antiTGF-β (ab66043, 1:200, abcam), anti-vinculin (v9131, 1:300, Sigma) were added into the samples and incubated overnight. On the following day, the primary antibody was removed and rinsed with PBS (3×), and it was incubated with the corresponding secondary antibody (1:1000) or the fitcphalloidinfor (Yeasen, 1:25) one h at 37 oC and counterstained with DAPI (Solarbio Science, China) for five minutes. All stained specimens were observed with the Nikon confocal laser microscope (Nikon, A1 PLUS, Tokyo, Japan). 2.11 Western blot analysis Different diameters of fibers were stuck to the 6-well culture plate as previously described. The fibroblast cells were seeded on the fibers (5×104 per well) for 1 day and 3 days, then cells were lysed by the protein extraction with supplemental protease. The protein concentration of lysed cells was quantified according to the BCA kit (Thermo, Rockford, IL, USA). The extracted protein was mixed with loading buffer and boiled and stored at -20 oC. In addition, 20 μl protein mixtures contain 40 μg total proteins were loaded to 12% polyacrylamide gel, separated at a voltage of 80 V. After 2 h, the separated protein was blotted on a PVDF membrane at a voltage of 120 V for 90 minutes. Subsequently, the membrane was blocked with 5% skimmed milk (BD/DicoTM) in TBST for 90 minutes. Followed by corresponding primary antibodies, and probes were added and incubation at 4 oC overnight. The primary antibodies include: anti-vinculin (v9131, 1:500, Sigma), anti-vimentin (ab92547, 1:1000, abcam), α-SMA (ab7817, 1:1000, abcam) and TGF-β (ab66043, 1:800, abcam), Gapdh (1:10000, bioworld), anti-integrin α1 (ab78479, 1:1000, abcam), anti-phospho-fak (ab4792, 1:800, abcam), anti-phospho-integrin β1 (ab5189, 1:800, abcam) and anti-phospho-paxillin (ab4832, 1:800, abcam). Later on, the corresponding secondary antibodies-goat anti-mouse IgG or goat antirabbit IgG (1:5000) conjugated with horseradish peroxidase (HRP) were added by combining the primary antibodies for 1 hour at room temperature. After this, the membrane was washed with TBST


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(3×), and signals were detected by ECL chemiluminescent agent and the results were captured via a ChemiDocXRS + Imaging System (Bio-Rad). Finally, the grey value of the target bands was analyzed by the Quantity One software, and GAPDH used for normalization. 2.12 Statistical analysis All data were expressed as mean ± standard deviations (SD). Statistical differences were performed by one-way analysis of variance (ANOVA), which was followed by Tukey’s test with GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA). For all tests, * p value ＜0.05, ** p value ＜0.01, *** p value ＜0.001.

3 Results and discussion 3.1 Characterization of Micro, Nano and Aligned fibers. Electrospinning is being widely employed for the preparation of 3D fiber matrix with the diameters ranging from nanometers up to micrometers with varied patterns 23. In this study, PCL as raw materials is chosen because it is biocompatible and its soft polymer stiffness is more close to extracellular matrix than inorganic materials for the improved cell adhesion. By adjusting the rate, concentration of PCL, all three kinds of fibrous scaffolds that interfaces with morphologies include randomly oriented microfibers, nanofibers and aligned nanofibers which were obtained with the use of the typical horizontal set up of electrospinning apparatus by focusing on different processing parameters24. As shown in Figure 1A-a&a’, microfiber with fibers diameter ranging from 1-2 μm are randomly oriented on the substrate through the SEM scanning results. The nanofibers matrix (Figure 1A-b&b’) is composed of comparatively thinner fibers with diameter of 200-400 nm. For the aligned nanofibers interface (Figure 1A-c&c’), the fiber with diameter of 200-400 nm is highly arranged with good alignment. Statistics analysis of average diameters upon each group were collected as shown in Figure 1B. Microfibers with average of 1.8 μm showed significantly increase of fiber diameter compared with other nanofibers (380 nm, *** p value < 0.001). Fiber morphology influences the mechanical properties of scaffolds. To clarify the effect of micro- and nano- structure on the mechanical properties of the electrospun fibrous matrix, the strainstress curves of different fibrous scaffolds were conducted and the results are shown in Figure 1C. Tensile curves of the randomly oriented fiber scaffolds go from elastic stage to plastic stage with strain hardening and necking phase compared to aligned fiber without strain necking phase but fractured immediately, suggesting randomly oriented fibers have multiple directions from the



randomly arrangement of fibers to reform the matrix after stretching. Aligned fibers only were arranged to one direction. Further elastic modulus showed the difference of the 3D fibrous matrix, and aligned fibered demonstrated the highest elasticity (0.019 MPa) and maximum tensile stress (1.44 MPa) due to the whole fibers aligned into one direction. Microfibers (elastic modulus 0.015 MPa, maximum stress 0.78 MPa) arranged in the middle between nanofibers (elastic modulus 0.007 MPa, maximum stress 0.42 MPa) and aligned fibers, showing that microfibers exhibited stronger tensile strength than nanofibers because of the larger diameters of the fibers with enhanced toughness.

Figure 1. (A) SEM images showing the surface morphology of different diameters and aligned electrospun PCL fibers. Scale bars: 10 µm. (B) Statistical results of average diameter in each group.


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(C) Tensile mechanical properties testing of microfiber, nanofiber, and aligned fiber fibrous scaffolds. And the statistics of modulus elasticity (5%-10%) and maximum tensile stress of different fibrous scaffolds. Associated contact angle experiments were further conducted to show the surface tension force by the 3D fibrous matrix. As shown in Figure 2A, all fiber scaffolds exhibited contact angle of water larger than 90o, suggesting the hydrophobic property of PCL. In particular, microfibers (Figure 2A-a) had water droplet with contact angle 131o, while nanofibers significantly reduced the contact angel to around 90o for both random (Figure 2A-b) and aligned nanofibers (Figure 2A-c). These results suggested that morphology of fibers (e.g. diameters) could influence the hydrophobicity of the fibrous scaffold. Nanofibers induced lower hydrophobic surface, resulting in increased surface energy. Microfibers revealed the minimum surface energy with the lowest surface tension force. In addition, statistical data of contact angle and contact distance ratio (/micro) were collected in Figure 2 B&C to reveal the significant difference of random micro- and nanofibers. In order to avoid the influence of inhomogeneity and mesh size of the fiber scaffold to contact angles, distribution of the diameter of the fibers and mesh size were plotted in Figure S1. It is observed that the fabricated PCL fiber demonstrated homogeneity with no significant difference of mesh size. When compared with random nanofibers and aligned nanofibers, they showed no obvious difference, indicating the arrangement of fibers has lower impact on the surface tension force of the fiber scaffold rather than diameters of fibers. Then typical AFM force-distance curve when the AFM tip retract from the fibrous scaffold after directly approaching to microfiber and nanofiber, respectively were obtained in Figure 2D. This negative force at the retract process of force-distance curve shown in the Figure 2D indicate the adhesion force. It is illustrated that the maximum adhesion force from microfiber is less than nanofiber. In addition, a comparison of the maximum adhesion is statistical analysis through several random locations on the fiber surface. The average adhesion force for microfiber is 27 nN compared to 40 nN for nanofiber (Figure 2E), indicating lower adhesion force with lower surface energy. Microfiber would have a lower drag tension force for following cell incubation, same results with the contact angle results.



Figure 2. (A) Hydrophilicity analysis of the PCL nanofibrous matrix with different diameter. (BC) Statistical results of contact distance ratio (/micro-) and contact angle in each group. (D-E) AFM height topography of samples of microfiber and nanofiber, and the statistical results of force of each group. 3.2 Morphology of 3T3 Fibroblast cell cultured on fibers Fibroblasts are the key players for orchestrating physiological wound regeneration. They embedded in the wound matrix that they build, meanwhile they sense the extracellular matrix. It is normally believed that the mechanical stress of the matrix would regulate the phenotype of fibroblast or myofibroblast. Thus here, fibroblast 3T3 cell lines was chosen to culture on the electrospinning fiber upon different diameters and oriented patterns with varied mechanical property.


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The general cell adhesion and spreading behavior on these fibers were studied by SEM. As the results shown in Figure 3, in contrast to aligned fiber (Figure 3A-c) with cells surface topology stretch along the fiber direction, both cells on random fibers of microfiber (Figure 3A-a) and nanofiber (Figure 3A-b) spread well after culturing for one and three days. This suggests that the cell behavior could be directed by the arrangement of the fiber scaffolds. Moreover, compared to the cell’s growing on the microfibers, the spreading areas of the cells culturing on the nanofiber were obviously increased with a clear trend to disperse themselves in all directions, indicating a significant cell deformation toward micro- and nanofibers. The statistical data of the cell area and aspect ratio were collected as presented in Figure 3B&C. These results further confirmed the enhancement of the cell area and aspect ratio (calculated from the ratio of cell length to width) with ** p value 10. 3.3 Cell spreading with upregulation of adhesion related cytoplasmic F-actin and vinculin Nest, actin stress fibers together with vinculin of 3T3 fibroblast were investigated through immunofluorescent staining. As shown in Figure 4A, the cell microfilaments F-actin (stained in green) and vinculin (stained in red) on day 3 further confirmed the morphology difference among these fiber scaffold. Vinculin, as a cytoplasmic actin-binding protein enriched in cell-cell and cellmatrix adhesions, can build the connection for cellular mechanic-transduction by regulating the polymerization and recruiting of adhesion actin remodeling proteins25. According to fibroblastmyofibroblasts development, the myofibroblast utilized specialized focal adhesions exhibiting specific high levels of vinculin.26 Here, obvious enhanced vinculin expressions with filamentous stress fibers were shown in spread cells grown on nanofibers (Figure 4A-b), indicating the cell adhesion were activated by nanofiber substrate with higher contract force from nanofiber scaffold to cells. Quantified data of the fluorescence intensity as shown in Figure S3 of cells on nanofiber is observed significant higher vinculin expression, followed by aligned fiber and microfibers, suggesting that random nanofiber induced more myofibroblast. This trigger of enhanced specialized focal protein from nanofiber further confirmed high surface tension from nanofiber scaffold, resulting in high mechanic-transduction from the fiber scaffold to cells. And quantified data of cell areas (from 913.4 μM2 to 1553.3 μM2) between microfiber and nanofiber) and aspect ratio (from 2.76 to 1.42) between microfiber and nanofiber) of cell cytoskeleton once again affirm the morphology of the spindle-shaped fibroblast cultured on nanofibers due to the enhanced tension stress from nanofiber matrix to cells. Further protein levels by western blot of vinculin from day one and day three respectively were collected as shown in Figure 4D&E. It demonstrates that with longer culturing on the fiber matrix, there existed an increase secretion of vinculin, indicating the enhanced vinculin expressions due to high attachment of cells to the fiber matrix. They followed the same changing pattern with immunofluorescent staining results on the day three. The vinculin expressions of cells on nanofibers showed the highest protein levels, indicating the highest tension strength of nanofiber scaffold to fibroblast cells. The overall results revealed a higher mechanical stress of nanofiber to the fibroblast, which leads to their transformation of spindle-shaped appearance of fibroblast with fibro nexus junctions such (e.g. vinculin) between the cells and the substrate. The mechanical force between cells and nanofibers further induced the vinculin activation


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and recruitment, and a large amount of vinculin was expressed for fibroblast grown on nanofibers. It is worth noted that, the vinculin of cells toward microfiber and aligned nanofiber have no significant difference may cause by dual factors including arrangement of fibers as well as fiber diameters.

Figure 4. (A) Immunofluorescent staining of Vinculin (red) and F-actin (green) after cell culture for 72 h. The clearer vinculin signal (red fluorescence) was detected when cells grown on the nanofiber. Scale bars: 50 µm. (B-C) Box-plot analysis of cell outlines and aspect ratio of cells on different fibers. Cell numbers n >10. (D-E) Western blot analysis of vinculin after cells culture on different supporting substrate for 24 h and 72 h. (F-G) The result of quantitative analysis of vinculin. *** P < 0.001, ** P < 0.01, * P

Controlling the Multiscale Network Structure of Fibers To Stimulate

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