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Non-Monotonic Self-Deformation of Cell Nuclei on Topological Surfaces with Micropillar Array Xiangnan Liu, Ruili Liu, Yexin Gu, and Jiandong Ding ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 23, 2017

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ACS Applied Materials & Interfaces

Non-Monotonic Self-Deformation of Cell Nuclei on Topological Surfaces with Micropillar Array Xiangnan Liu, Ruili Liu, Yexin Gu, Jiandong Ding1 State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai 200433, China

Keywords: Nuclear deformation; Polymeric biomaterials; Cell nucleus; Micropillar array; Mechanotransduction

Corresponding author. Tel.: 86 21 65643506. Fax: 86 21 65640293. E-mail address:[email protected] (JD Ding). 1

ACS Paragon Plus Environment


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Abstract Cells respond to the mechanical signals from their surroundings and integrate physiochemical signals to initiate intricate mechanochemical processes. While many studies indicate that topological features of biomaterials impact cellular behaviours profoundly, little research has focused on the nuclear response to a mechanical force generated by a topological surface. Here, we fabricated a polymeric micropillar array with an appropriate dimension to induce a severe self-deformation of cell nuclei and investigated how the nuclear shape changed over time.

Intriguingly,

the

nuclei

of

mesenchymal

stem

cells

(MSCs)

on

the

poly(lactide-co-glycolide) (PLGA) micropillars exhibited a significant initial deformation followed by a partial recovery, which led to an “overshoot” phenomenon. The treatment of cytochalasin D suppressed the recovery of nuclei, which indicated the involvement of actin cytoskeleton in regulating the recovery at the second stage of nuclear deformation. Additionally, we found that MSCs exhibited different overshoot extents from their differentiated lineage, osteoblasts. These findings enrich the understanding of the role of the cell nucleus in mechanotransduction. As the first quantitative report on non-monotonic kinetic process of self-deformation of a cell organelle on biomaterials with unique topological surfaces, this study sheds new insight on cell-biomaterial interactions.


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1. INTRODUCTION Cells sense their physical environment and adjust to variable mechanical influences by converting biophysical and biochemical cues into intracellular signals.1-4 Materials with characteristic structures and properties offer a well-defined microenvironment to investigate cell-environment interactions.5-10 Valuable guidance has been provided for the development of scaffolds for tissue engineering and regenerative medicine.11-15 Topologically patterned surfaces have exerted significant influence on the behaviors of adhered cells including deformed cell shapes.16-22 While most of the reports focus on the overall responses of cells, little attention has been paid to the subcellular response of an organelle to topological surfaces. As the largest and stiffest organelle in a cell, nucleus plays an important role in regulating cell mechanics.23 Recently, topological surfaces with micropillar arrays of appropriate dimensions were found to trigger pronounced nuclear deformation.24-30 Such amazing and sustained distortion of cell nuclei was not easily achieved by previous nuclear deformation inducing techniques such as micropipette aspiration31 and atomic force microscope.32 External forces from the topological surfaces can be transmitted from the adhesion molecules dispersed at the cell surface to the cell nucleus, which is physically coupled to the cytoskeleton via the so-called “linker of nucleoskeleton and cytoskeleton (LINC)” complex.33-34 Nuclear mechanotransducton cascades once the nucleoskeleton responds to the forces that are transmitted from the LINC complex.35-36 In that regard, cell nucleus is considered to be mechanosensitive. Inside the nucleus, these forces change nuclear structures and functions, which in turn contribute to the sensitivity of the whole cell to mechanical



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forces and even alter transcriptional regulation.37 For example, the deficiency of Lamin A/C leads to defective nuclear mechanics in response to mechanical strain and impair mechanically activated gene transcription.38 Hence, the nucleus is responsive to the “outside-in” mechanical forces from the surroundings, and also affects processes inside the nucleus to activate “inside-out signaling”. The stem cells and differentiated cells show different nuclear mechanics, which are determined by nuclear lamina and chromatin.39 Discher’s group found that nuclei in undifferentiated stem cells deformed more readily than those in differentiated cells using micropipette aspiration.40 These evoked our interest to explore how the nuclear shapes of stem cells, differentiated cells and differentiating cells dynamically respond to topological surfaces with micropillar arrays. Here, we fabricated micropillar arrays of poly(lactide-co-glycolide) (PLGA) with indicated dimension in Figure 1 in order to detect the kinetic process of self-deformation of cell nuclei on topological biomaterials. We studied quantitatively the nuclear deformation of mesenchymal stem cells (MSCs) and osteoblasts in a time-dependent manner. The fractions of deformed nuclei and average shape index (SI) were calculated to quantify the nuclear shapes as a function of time. We also explored a more complicated case of stem cells during differentiation by monitoring the change of nuclear shapes of cells in osteogenic medium. A non-monotonic kinetics of the self-deformation of cell nuclei on topological surfaces with micropillar arrays was found in all of the examined cases.


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Figure 1. Schematic presentation of the nuclear deformation on a micropillar array. (a) The nucleus of a cell deforms when the cell is seeded on an appropriate micropillar array, which is fabricated from a silicon wafer template with micropit. (b) SEM images of the PLGA micropillar array with a height of about 7 µm, a pillar spacing of 6 µm and a pillar size of 3 × 3 µm2.

2. MATERIALS AND METHODS

2.1. Fabrication and Observations of Micropillar Array. The fabrication process of the model micropillar surface began with the preparation of a PLGA (Purac Inc., Netherlands) solution in dichloromethane. The purchased PLGA has a lactide/glycolide composition of 85/15 and number-average molecular weight of 3.59×105. The PLGA solution was casted onto a silicon wafer template with a customized micropit array. After dried at room temperature for 48 hours, the PLGA films with a micropillar array on the surface were carefully removed from the silicon mold through vertical pulling and stored in a desiccator under vacuum until use. As the control, smooth PLGA films were obtained through an unpatterned silicon wafer template with the same method. The micropillared film was



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sputter-coated with gold and observed by scanning electron microscopy (SEM, VEGA TS5136MM). The electric voltage was set as 20 kV during the morphological observation.

2.2. Cell Harvesting and Culture. MSCs were obtained from 7 day old neonatal Sprague Dawley (SD) rats. The isolated marrow cells were seeded in culture flasks and cultured in low-glucose Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). After 48 hours, the growth medium was refreshed to remove the non-adhesive cells. The stem cells were subcultured upon almost confluence. The second-passage MSCs were used in the subsequent experiments. The osteoblasts were obtained from the osteogenic differentiation of MSCs. Briefly, the second-passage MSCs were exposed to osteogenic medium for 7 days. We prepared the osteogenic medium by addition

of

FBS

(10%,

Gibco),

ascorbic

acid-2-phosphate

(50

µM,

Sigma),

β-glycerophosphate (10 mM, Sigma) and dexamethasone (100 nM, Sigma) into high-glucose DMEM (Gibco). The medium was refreshed on the third and fifth days of induction. In order to inhibit cell proliferation, aphidicolin (0.5 µg/ml, Sigma) was added during the induction period.

2.3. Cell Seeding and Culture on Various Substrates. After sterilization with 75% alcohol for 45 min, the PLGA films with or without micropillar arrays were transferred in 12-well tissue culture plates (TCPs) and then rinsed thoroughly with phosphate buffered saline (PBS, pH 7.0 to 7.2). MSCs and osteoblasts were seeded on the PLGA films at a density of 5 × 103 cells per cm2 and cultured in growth medium in a humidified incubator at 37 °C with 5% CO2 atmosphere. In another group, MSCs seeded on the topological substrates were cultured in the osteogenic media for osteogenic differentiation. Cells were incubated up


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to 7 days and analyzed at 1, 2, 4, 8, 16, 24, 72, 120 and 168 hours. Media were renewed every 2 or 3 days. Aphidicolin was added to control the cell density. In order to investigate the contributions of actin filaments to the nuclear responses of MSCs on topological surfaces, a cytoskeleton disruptive agent cytochalasin D (Sigma) was employed to depolymerize actin filaments. In detail, MSCs of the second passage were digested, centrifugalized and resuspended in growth medium which contained 0.25 µg/mL cytochalasin D. Then MSCs were seeded at density of 5 × 103 cells per cm2 on micropillar array and cultured for 24, 72, 120 and 168 h. Media were refreshed every 2 or 3 days.

2.4. Staining and Observations of Cells. The cells cultured for 1, 2 and 4 hours were rinsed gently only once, because cells might not adhere very well on micropillars during these periods. Then the remaining cells were treated with Hoechst 33342 (10 µg/ml, Sigma) for 10 min to label nuclei, and photographed quickly under an inverted microscope (Axiovert 200, Zeiss) mounted with a charge coupled device (CCD, AxioCam HRC, Zeiss) for further statistical analysis. For the cells cultured for 8, 16, 24, 72, 120 and 168 h in growth medium or osteogenic medium, they were carefully rinsed with PBS and fixed with 4% paraformaldehyde for 10 min. The cells were permeabilized with 0.1% Triton X-100 for 10 min and then rinsed with PBS. To label the filamentous actins (F-actins), the preprocessed cells were treated with 1 µg/ml phalloidin-tetramethyl rhodamine B isothiocyanate (phalloidin-TRITC, Sigma) for 30 min. Subsequently, nuclei were stained with Hoechst 33342 (Sigma). After rinsing with Milli-Q water, we observed the cells and captured the images under the microscope. The fluorescently stained MSCs were also observed under a confocal laser



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scanning microscope (LSM 710, Zeiss) for a further morphological study in Z-direction. Serial cross-sectional images were obtained and image-J 1.48v software (freely available at http://www.imagej.nih.gov/ij/) was used to outline the profile of cell nuclei in each image to get the ordinate values of center-mass. The heights of recovered nuclei were normalized to the mean values of the deformed nuclei group. At least 40 cells were analyzed from more than 15 confocal images for each group. 2.5. Statistical Analysis. We employed shape index (SI), which is defined as 4πS/l2, to quantify the deformation extent of cell nuclei. Here, S and l denote the spreading area and perimeter of a cell nucleus, respectively. S and l of each nucleus were measured from the acquired nuclear profile through image-J. At least 180 nuclei of cells were randomly collected and analyzed in each group at each time point. Independent experiments (n = 3) were carried out to determine the average and standard deviation in the analysis of nuclear deformation. One-way ANOVA tests were performed with p < 0.05 considered as a statistically significant difference between two groups.

3. RESULTS 3.1. Nuclear Responses of MSCs on Micropillars in Growth Medium. Micropillar arrays were prepared by using a silicon wafer template with micropits. According to the SEM images (Figure 1b), a micropillar array with a height of about 7 µm, a pillar spacing of 6 µm and a pillar size of 3 × 3 µm2 was acquired. Such a dimension of the pillars can induce a severe change of nuclear shape, as schematically demonstrated in Figure 1a. The cell was deformed as well, and a large part of cytoplasm fell into the space between the micropillars.


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The nuclear shape changed dynamically. The MSCs cultured on micropillars were fluorescently stained to investigate the nuclear responses of cells in growth medium. Representative confocal fluorescence images of actin cytoskeletons and nuclei of the cells cultured for 24 and 120 h are shown in Figure 2a. Some F-actins were concentrated along the micropillar array and most of the nuclei deformed between the micropillars at 24 h. At 120 h, cells adopted a more spread morphology on micropillars, and most of the cell nuclei were no longer located between the micropillars instead stretched on the top of pillars. The indicated SI values were much larger than those at 24 h, demonstrating that the initially deformed nuclei partially recovered on micropillars along with time. The distribution of nuclear SI was obtained for each time point, with results shown in Figure 2b. Interestingly, the peaks moved to the left (24 h) and then back to the right (120 h). In accordance with this, the average SIs presented an abrupt drop in less than 24 hours and slowly ascended later on, as shown in Figure 2c. The distributions of SI corresponding to each data point in Figure 2c are displayed in Figure S1, and the controlled cell densities by aphidicolin are indicated in Figure S2.



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Figure 2. Nuclear responses of MSCs in growth medium along the time. (a) Representative confocal fluorescence micrographs of cells with F-actins stained in red and nuclei in blue at indicated time points


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after seeding cells on the micropillar array. Shape index as an indicator of deformation degree is defined as SI = 4πS/l2, where S and l denote the cell area and perimeter, respectively. Some values of SIs are indicated beside the corresponding cell nuclei in the lower row. (b) Some typical distributions of SI on micropillars and smooth films at indicated time points. (c) Fractions of deformed nuclei and average SIs as a function of culture time. We chose 1, 2, 4, 8, 16, 24, 72, 120 and 168 hours to carry on statistics of nuclei of MSCs. At least 450 cells were assessed at each time point on micropillared and smooth films.

The self-deformation of cell nuclei on the micropillar array could be divided into two stages, the initial severe deformation at the first stage and the partial recovery at the second stage. Comparison between the equilibrium SIs between micropillared and smooth films illustrates that the net result is still a significant self-deformation of cell nuclei. The recovered nuclei of MSCs on micropillars were further evaluated by confocal microscopy. A typical image is presented in Figure 3a, including top-down sections with both deformed nuclei and recovered nuclei. The height profiles along the Z direction clearly show that the position of recovered nuclei is higher than that of the deformed nuclei, as schematically presented in Figure 3b. The relative heights of cell nuclei stretched on the top of the pillars are about 3 µm higher than those of cell nuclei deformed between the pillars, with the statistical results shown in Figure 3c. The relative locations of nuclei and micropillars determined nuclear deformation or recovery in the micropillar environment. Besides, the aspect ratios (ARs) of partially recovered nuclei were smaller than that of the deformed nuclei, as shown in Figure S3.



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Figure 3. Comparison of the relative heights of deformed nuclei and partially recovered nuclei of MSCs

on micropillar arrays. (a) Confocal microscopic image of MSCs after 72 h culture in growth medium on micropillars. F-actins (red) and cell nuclei (blue) were visualized through fluorescent staining. The top and right fluorescence images depict the corresponding height profiles of the top-down sections along the yellow lines. Both deformed nuclei and partially recovered nuclei with different heights are shown in the cyan rectangle. (b) Schematic of the shapes and positions of cell nuclei deformed between the pillars and stretched on the top of the pillars. (c) Statistical result of the relative heights of recovered nuclei normalized by the mean values of the deformed nuclei group. At least 40 cells were analyzed from more than 15 confocal images for each group. A significant difference is marked with “***” (p < 0.001).

3.2. Treatment of MSCs on Micropillars with Cytochalasin D. In order to test whether actin filaments played an important role in the nuclear deformation of MSCs on micropillar arrays, we cultured the cells in growth medium containing cytochalasin D for 24, 72, 120 and 168 h. Like the first stage of initial nuclear deformation without cytochalasin D, cell nuclei significantly deformed at 24 h after the actin filaments were disrupted. Yet unlike the control group without cytochalasin D, the nuclear deformation in the group with cytochalasin D sustained on micropillars up to 120 h (Figure 4a) with the indicated values of SI less than 0.8. The F-actins were diffused and accumulated around the micropillars more obviously at 120 h.


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Figure 4. Effect of cytochalasin D on the kinetic process of nuclear deformation of MSCs in growth

medium. (a) Fluorescence images of MSCs in growth medium on micropillars with the treatment of cytochalasin D for 24 and 120 hours. F-actins (red) and cell nuclei (blue) were visualized through fluorescent staining. The numbers in the second row indicate the corresponding shape indexes of nuclei with smaller values referring to more significant nuclear deformation. (b) Fractions of deformed nuclei and average SI of cell nuclei on micropillars with and without the treatment of cytochalasin D, indicating the maintenance of self-deformation of cell nuclei and disappearance of nuclear recovery of cells on micropillars after suppression of formation of actin filaments by cytochalasin D.



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We further compared the fractions of deformed nuclei and average SI between cells with and without the treatment of cytochalasin D, as demonstrated in Figure 4b. The distributions of SI with the treatment of cytochalasin D at different time points are presented in Figure S4. All of these data support that while the treatment of cytochalasin D did not inhibit occurrence of nuclear deformation at the first stage, the otherwise nuclear recovery at the second stage of nuclear deformation was significantly suppressed when the actin filaments were disrupted after the addition of cytochalasin D.

3.3. Nuclear Responses of Osteoblasts on Micropillars in Growth Medium. Osteoblasts, as another cell type, were also examined in growth medium. Typical fluorescence images of actin cytoskeletons and cell nuclei at 120 h are presented in Figure 5a. The stained F-actins were much brighter around the pillars in some areas. Most of the nuclei deformed severely among the micropillars, as seen from the SI distribution graphs of nuclei (Figure 5b). The distribution regions less than 0.8 were dominant at both 24 h and 120 h on micropillars compared with that on smooth films. Similar to MSCs, the deformation trends in Figure 5c changed dramatically at the initial time after seeding osteoblasts. The distributions of SI corresponding to each data point in Figure 5c are displayed in Figure S5. The kinetic trajectory of the nuclear deformation also exhibited two stages, rapid initial deformation and slow partial recovery, as shown in Figure 5c. However, due to relatively less recovery at the second stage, the overshoot during the process of nuclear deformation for osteoblasts is less significant than the MSCs on micropillars.


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Figure 5. Nuclear responses of osteoblasts in growth medium along the time. (a) Typical fluorescence micrographs of cells with F-actins stained in red and nuclei in blue at 120 hours after seeding cells on micropillars. (b) Some typical distributions of SI of osteoblasts in growth medium on micropillared and smooth films at indicated time points after seeding cells. (c) Fractions of deformed nuclei and average SIs as a function of culture time for osteoblasts. At least 450 cells were assessed at each time point on micropillared and smooth PLGA films.



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3.4. Nuclear Responses of MSCs on Micropillars in Osteogenic Medium. To further explore the nuclear responses of cells on micropillars, induction of MSCs towards osteoblasts on micropillars was carried out by culturing the cells in the osteogenic medium. The images with actin cytoskeletons and nuclei stained at 120 h are shown in Figure 6a. While the cells spread well on micropillars, most of the nuclei deformed severely. The SI distribution graphs of nuclei in Figure 6b indicated that peak positions located at more than 0.8 at 1 h, less than 0.8 at 24 h, both more than and less than 0.8 at 120 h on micropillars resulting in double peaks. The fraction of deformed nuclei dramatically rose at initial time, then gently decreased along with time and tended to be constant in the end, as shown in Figure 6c. The curve of average SI of cell nuclei also underwent an abrupt fall and following a gentle rise. It is interesting that the average SI of cell nuclei dropped again after inducing the MSCs for about 72 h. So here, the whole nuclear deformation process includes three stages with two overshoots. Stages 1 and 2 are still initial self-deformation and partial recovery of cell nuclei; the third stage might arise from the change of cell type during stem cell differentiation. The distributions of SI corresponding to each data point in Figure 6c are displayed in Figure S6. We tried to figure out the reason by comparing the average SI of deformed nuclei of MSCs in growth medium and osteogenic medium on micropillars (Figure S7). Significant difference of average SI of deformed nuclei was found between culture in growth medium and osteogenic medium at 5 or 7 d. In other words, the nuclear deformation of cells is much more severe in osteogenic medium than in growth medium. Meanwhile, the differentiated cell type (osteoblast) has more severe nuclear deformation than undifferentiated MSCs, which


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contributes the third stage of the nuclear deformation during the osteogenic differentiation of the stem cells.

Figure 6. Nuclear responses of MSCs in osteogenic medium along the time. (a) Representative fluorescence micrographs of cells with F-actins stained in red and nuclei in blue at 120 hours after seeding cells. (b) Distributions of SI of MSCs in osteogenic medium on micropillared and smooth films at



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indicated time points. (c) Fractions of deformed nuclei and average SIs as a function of induction time for MSCs. At least 450 cells were assessed at each time point on both micropillared and smooth PLGA films.

4. DISCUSSION

The interactions between cells and materials with topological surface can lead to changes of cellular and subcellular geometry.41-47 Microstructured surfaces prove to be an ideal platform to induce detectable nuclear deformation since the physical dimension of a nucleus is commonly on the microscale. Nuclear deformation occurs also in vivo. For example, it was found that cell nuclei deformed during migration through narrow extracellular matrix fibers48 or intravasation across blood vessel.49 Although a series of microstructured surfaces with various spacings,27-28 pillar sizes28 and heights29 were applied to investigate nuclear deformation of different cell types in recent years, some very fundamental questions remain to be explored, for instance, how nuclear deformation is initiated at early stage and how cellular nucleus respond to the topological substrates in a time-dependent manner. In the present study, a PLGA substrate with the micropillar structure was prepared to examine the nuclear responses of cells, as schematically presented in Figure 1. To quantify the response of nuclear shape at each time point, we used the fraction of deformed nuclei and the average SI. A cell nucleus with SI less than 0.8 is suggested as a deformed nucleus according to our statistics of many distribution curves. The nuclei of MSCs got to deform at the moment that the cells attached on the micropillar arrays. At 2 h, the fraction of deformed nuclei was already up to about 50% (Figure 2). The deformed nuclei recovered later on after 16 h and the relative height of partially recovered nuclei was about 3 µm higher than the deformed nuclei of cells at 72 h (Figure 3).


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Micropillar-induced response of nuclear shape is instantaneous and reversible. Overall, the process of nuclear recovery is much slower than the initial nuclear deformation. The nuclear recovery is partial instead of complete, for the asymptotic values at the ends of the curves of deformed nuclei fractions and average SI on micropillars were far away from those on smooth films. The observation of nuclear recovery implies that the deformed nuclei broke through the confinement of micropillars and stretched on the top. To figure out how the nuclear recovery happened, we treated the cells with cytochalasin D to depolymerize the actin filaments and then observed the nuclear shape. As shown in Figure 4, disruption of actin filaments didn’t prevent the cell nuclei from deforming. Similarly, nuclear deformation using micropipettes could still remain even after treatment of cells with cytochalasin D.50 Thus the first stage of nuclear deformation on micropillars seems more mechanical than biological. But the deformed nuclei no longer recovered in our experiment if the actin filaments were disrupted, which illustrated that actin cytoskeleton took part in providing the driving force to pull the deformed nuclei out of the confined space between the micropillars in the case without cytochalasin D. If we took the partial nuclear recovery into consideration, the phenomenon of self-deformation of cell nuclei on micropillar arrays could be described as “overshoot”. In physics, overshoot refers to a kinetic response process in which a signal or function exceeds its final or steady-state value. In our research, fractions of deformed nuclei as a function of time exhibited a positive overshoot, because the initial percentage of deformed nuclei



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exceeded the final, steady-state value. Meanwhile, time-dependent SIs exhibited a negative overshoot. The nucleus appears to firstly respond to the extracellular environment or mechanical stimulus in a manner that mirrors the surroundings. For instance, the nuclear shape extended when the cell elongated and spread.51 When the cells were seeded on stiff substrates, the expression of lamin A increased and the nucleoskeleton also became stiffer.52 Here, the deformed nuclei showed a compliance with micropillars. Accompanied with cell adhesion, most of the cytoskeleton inserted between the micropillars, and so did the nuclei. The external force from the micropillar arrays acted on the cells and directly triggered biochemical signaling network, which connected extracellular matrix, cytoskeleton and nuclei all together. So the nuclear deformation induced by the external stimulus occurred very rapidly. This conclusion was confirmed in other research systems using micropipette aspiration and pyramidal AFM.32,50 It has even been found that the nuclear deformation could also result in condensation of chromatin and change of gene expressions.37,53 The “inside-out” signals from the nuclei were conducted to cytoskeleton network through LINC complex, and the action filaments exerted a pulling force upon the deformed nuclei in respond to the nuclear deformation. Then the deformed nuclei rebounded from the micropillared films and reverted to typically spheroidal or ellipsoid as much as possible. Hence, the final nuclear shape depended on both the initial nuclear deformation at the first stage and the partial nuclear recovery at the second stage, as schematically presented in Figure 7a.


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Figure 7. A summarized presentation of the overshoot phenomenon in deformation of cell nuclei on micropillars. (a) The nucleus of a cell seeded on micropillars undergoes significant self-deformation with an overshoot and then a rebound response. Final nuclear shape is derived by the superimposition of the opposite effects of nuclear deformation and partial recovery. (b) Comparison of nuclear deformation of MSCs and osteoblasts on micropillars. (c) In growth medium, the cell nuclei on the micropillar array undergo a non-monotonic kinetic process with an overshoot between two stages. MSCs in the osteogenic medium exhibited also the further nuclear deformation due to change of the cell type in stem cell differentiation, resulting in the third stage.

Nuclei of osteoblasts deformed faster than MSCs but only slight nuclear recovery was displayed (Figure 5). Most of deformed nuclei remained for as long as 7 days. For osteoblasts, the overshoot phenomenon is not as obvious as for MSCs, and the final nuclear deformation is more significant than that for MSCs, as presented in Figure 7b.



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We were then curious about how a dynamically differentiating cell responded to the micropillar array. Hence, MSCs on micropillars were induced in the osteogenic medium to observe the change of nuclear shape during the induction period, with the results shown in Figure 6. For convenience of comparison, experimental curves that quantified the dynamic change of nuclear shape were merged together under several scenarios including MSCs in growth medium, osteoblsts in growth medium and MSCs in osteogenic medium (Figure S8). MSCs in osteogenic medium exhibited three stages with two overshoots of their time-dependent nuclear deformation. While the first overshoot with an abrupt fall and then a mild rise of SIs is due to the partially nuclear recovery, the second overshoot comes from the commitment of the stem cells towards the osteoblastic phenotype leading to the further decrease of SIs. A summarized presentation of the dynamic changes in nuclear shape is illustrated in Figure 7c. Based on the observations, we can speculate that the nucleus act as the hub of mechanotransduction and respond to the extracellular environment in an overshoot manner by altering its compliance and resistance to micropillars. The “outside-in” signals result in nuclear compliance to micropillars with deformation, and the “inside-out ” signals lead to nuclear resistance to micropillars with recovery.

5. CONCLUSIONS The kinetic process of the nuclear deformation of cells on the PLGA micropillar array prepared by us contains basically two stages with an overshoot. While the first stage (rapid and severe initial self-deformation) is more mechanical, the second stage (relatively slow partial recovery) is highly biological since actin filaments are necessarily involved in such a


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recovery stage. The overshoot of MSCs in growth medium was more obvious than that of osteoblasts in growth medium, and more significant nuclear deformation was presented in osteoblasts at last. The most complicated kinetics occurred in the case of MSCs in the osteogenic medium, resulting in three stages with two overshoots. The new last stage exhibited further nuclear deformation due to change of cell type during differentiation of the stem cells. So, the geometry of cell nuclei responds to the micropillar array in an overshoot manner. The finding of the non-monotonic kinetic process of significant self-deformation of a cell organelle like the nucleus on the topological surface deepens the understanding of cell-biomaterial interactions, which may be taken into consideration in rational design of new biomaterials.

ASSOCIATED CONTENT Supplementary Information Time-dependent distributions of nuclear SI of MSCs, cell densities of MSCs on micropillars, AR of deformed nuclei and partially recovered nuclei of MSCs, distributions of nuclear SI of MSCs with the treatment of cytochalasin D, distributions of nuclear SI of osteoblasts, distributions of nuclear SI of MSCs under differentiation, average SIs of deformed nuclei of MSCs cultured in growth and osteogenic medium, comparison among all of the three cases in our experiments. The Supporting Information is available free of charge on the ACS Publications website at DOI: ***. AUTHOR INFORMATION



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Corresponding Author 1

E-mail: [email protected]. Phone: 86 21 65643506. Fax: 86 21 65640293.

ORCID Jiandong Ding: 0000-0001-7527-5760 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was financially supported by National Science Foundation of China (grant no. 51533002, 51503043, and 51603045), and State Key Project of Research and Development (grant No. 2016YFC1100300).


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