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Effects of cyclic fluid stress at different frequencies on behaviors of cells incubated on titanium alloy
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Effects of cyclic fluid stress at different frequencies on behaviors of cells incubated on titanium alloy Xing Lei a, b, Hao Wu a, Yue Song a, Bin Liu a, Shuai-Shuai Zhang a, Jun-Qin Li a, Long Bi a, *, Guo-Xian Pei a, ** a b
Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, China Department of Orthopedic Surgery, Linyi People’s Hospital, Linyi, 276000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 October 2019 Accepted 12 November 2019 Available online xxx
The orthopedic external fixation is always in dynamic mechanical environment with the somatic movement. We used a self-designed mini oscillator to simulate this condition by providing the reciprocating cyclic fluid stress, and observed the behavioral responses of fibroblasts implanted on titanium alloy plane to the stress at different frequencies, including 0.2 Hz, 0.6 Hz, and 1.0 Hz. We found that the cell angle, shape index and expression of vinculin were mostly biphasic-dependent with the increase of frequency, with peaks at 0.6 Hz. Whereas the cell area, expression of Col-I and a-SMA were mainly affected by the 1.0 Hz stress. Interestingly, 1.0 Hz stress also promoted Col-I expression of bone marrow mesenchymal stem cells (BMSCs), although it did not increase a-SMA. These results reveal that 0.6 Hz stress improves the alignment, polarity and adherence of fibroblasts on titanium alloy substrates, thus improving the sealing of implants; the 1.0 Hz force activates the differentiation of fibroblasts into myofibroblasts and increases collagen produced by stem cells, which probably cause the formation of fibrous capsules around implants. © 2019 Elsevier Inc. All rights reserved.
Keywords: Cyclic fluid shear stress Fibroblasts BMSCs Alignment Vinculin Differentiation
1. Introduction Many studies have done a lot of work on the surface modification of percutaneous devices to promote the integration of the materials and skin and prevent the pin tract infection [1], but neglected the influence of external mechanical environmental factors on the implant sealing. Especially in orthopedics, implants, such as external fixation, are always in dynamic environment with the somatic movement, which is different from the static stress environment of denture in stomatology. Inspired by the above idea, in this work, we self-designed a mini lab oscillator to provide a reciprocating fluid shear force to simulate the mechanical environment sensed by external fixation, and tried to reveal the behavioral responses of the major participants in skin tissue integration, fibroblasts [2] and stem cells [3], to dynamic mechanical forces. In terms of frequency setting, we took the walking speed of
* Corresponding author. Department of Orthopedics, Xijing hospital, Fourth Military Medical University, 15 changle West street, Xi’an, 710032, China. ** Corresponding author. E-mail addresses: [email protected] (L. Bi), [email protected] (G.-X. Pei).
human lower limbs as a reference. The walking frequency of normal adults is close to 1 Hz (0.97 ± 0.04 Hz) in a gait cycle [4], which is defined from the initial foot contact to the following ipsilateral initial contact. For the elderly or traumatized, the frequency usually becomes smaller (Fig. S1). So in this work, the maximum cycle frequency of the oscillator is set to 1.0 Hz, followed by 0.6 Hz and 0.2 Hz as transitions, with static culture as a control. We observed the morphological responses of fibroblasts planted on titanium alloy plane under fluid stress of different frequency, such as cell angle, polarity, area and number. Theoretically, if the cell alignment is better and the polarity is greater, the extracellular matrix (ECM) produced will be more parallel and ordered, and the sealing of skin-implants will be more good [5]. In addition, we also examined the expression of adhesion and differentiation markers in fibroblasts, including fluorescence intensity, gene or protein expression of vinculin, Col-I and a-SMA, to evaluate the effects of different frequency stresses on the sealing strength of skin-implants [6] and tissue repair ability [7,8]. Because stem cells are involved in the epidermal regeneration and wound healing [9], this study also explored what role stem cells played in skin-implants sealing under dynamic fluid forces by using bone marrow mesenchymal stem cells (BMSCs).
https://doi.org/10.1016/j.bbrc.2019.11.070 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: X. Lei et al., Effects of cyclic fluid stress at different frequencies on behaviors of cells incubated on titanium alloy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.070
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2. Materials and methods
2.6. Immunofluorescence
2.1. Samples preparation and surface characterization
Immunofluorescence was performed by using our previously experimental protocol [11]. The antibodies include: vinculin (Sigma), types I collagen (Col-I) antibody (Abcam),a-SMA (Sigma), goat anti-mouse-FITC (Sigma). F-actin was stained with phalloidinTRITC (Cytoskeleton) and the nucleus with DAPI (Thermo). All specimens were then examined under a fluorescence microscope (Carl Zeiss) at low magnification (5 ) or a laser scanning confocal microscopy (Nikon) at high magnification (40 ). The fluorescent images were quantitatively analyzed using NIH Image J software.
Square TC4 titanium alloy (hereinafter referred to as Ti-A) sheets (10 mm 10 mm length, 1 mm thick) were purchased from Shenzhen Chuangyifu Metal Materials Co., Ltd. The Ti-A substrates were mechanically polished to achieve smooth and mirror like surfaces. All samples were cleaned and sterilized with 90% ethanol for 4 h, and washed with sterile PBS before plating the cells. Surface topography was imaged by LaB6 scanning electron microscopy (SEM) (Tescan) and quantified by atomic force microscopy (AFM) (Agilent). Water contact angle measurements were investigated by JC2000DM contact angle measuring instrument (Zhongyi Kexin Technology Co., Ltd.). 2.2. Cell culture NIH-3T3 (mouse embryonic fibroblast cell line, Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and mouse bone marrow-derived mesenchymal stem cells (BMSCs, which were isolated from the femur bone marrow of 2-week-old mice. See our previous published research for more details [10]) were cultured in DMEM with 10% FBS. NIH-3T3 (2 104/ml) or BMSCs (1 104/ml) were seeded onto the Ti-A or groove substrates and incubated for 8 h in 24-well microplates. Cellular analyses were carried out at 48 h after samples were transferred from microplates to f100mm cell culture dishes. 2.3. Biocompatibility and cell viability assays The biocompatibility of Ti-A sheets was tested by examining the growth of NIH-3T3 cells using the live/dead viability assay. Square glass pieces with the same size as the Ti-A were used as negative control substrates. NIH-3T3 cells were seeded at 1.2 104 cells per sheet. The cells were double stained with Calcein-AM (MedChemExpress) and EthD-1 (Sigma-Aldrich) combined solutions on each substrate for 12, 24 and 48 h. Images were obtained using an Axio Observer A1 optical microscope (Carl Zeiss) equipped with fluorescence light source and filters. The number of live/dead cells were counted and analyzed by using NIH Image J software. 2.4. Construction of fluid shear stress models at different frequencies In order to simulate the mechanical shear stress around the external fixator during muscle flexion and extension in vivo, we designed a mini lab oscillator which can provide reciprocating fluid shear force to act on cells (Video S1). Two f100mm cell culture dishes were placed in parallel on the oscillator, with six Ti-A sheets implanted in each dish. The oscillator can be placed in a cell culture incubator to provide power without affecting the cell culture environment (Video S2). Three grades of 0.2, 0.6 and 1.0 Hz were selected as the experimental groups, and the amplitude is 2 cm; cells cultured in static state were used as a control. The oscillator worked 18 h/day.
2.7. Morphological analysis At low magnification, nine visual fields per sample were randomly captured for morphological analysis. Cell orientation was defined as the angle between the long axis of cells and the direction of the shear force; cell shape index (CSI) was based on the ratio of the long axis to the short axis. The inclusion criteria of the above two indicators: (i) Cell outline is clear; (ii) Showing cell polarity. Exclusion criteria: (i) intercellular fusion leads to unclear contour; (ii) more than 2 branches; (iii) obviously curved cells; (iv) binuclear or multinucleated cells. Besides, because of the very small proportions in each group, mononuclear spherical cells were not calculated, either. The number of cells was quantified by counting nuclei. The cell area and the fluorescence intensity of F-actin were also measured in this work. At high magnification, more than five images per specimen were randomly visualized for morphological analysis. FAs were quantified using an anti-vinculin antibody, including fluorescence intensity, FA area and number per cell, and FA area per focal contact point. Differentiation from fibroblasts to myofibroblasts was determined by analyzing fluorescence intensity of Col-I and a-SMA [12,13]. All images were analyzed with NIH Image J software. 2.8. RNA purification, semi-quantitative RT-PCR (sqPCR) and quantitative RT-PCR (qPCR) qPCR was performed using standard protocol as our previously described [11]. Total RNA from the treated cells was extracted using the E. Z.N.A.® Total RNA Kit I (Omega). Total RNA was quantified with a spectrophotometer. Reverse transcription was performed using the two-step PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). sqPCR was performed using Taq™ PCR mix (Takara). The 2 DDCt method was used for data analysis, and mRNA expression was shown relative to control 24 h group. The primers, designed by Takara Biotechnology Co., Ltd., are shown in Table S1. 2.9. Western blotting WB analysis was performed using a standardized protocol as described previously [10]. The antibodies include: Col-I antibody (Abcam), a-SMA (Sigma), b-actin (Affinity), and HRP-linked goat anti-mouse IgG (H þ L) (Affinity). 2.10. Statistical analysis
2.5. SEM After 48 h of fluid force acting on NIH-3T3 cells seeded onto the Ti-A substrates, cell morphologies were assessed via SEM (Tescan). Briefly, the samples were fixed with 2.5% glutaraldehyde at 4 C for 4 h, dehydrated through a graded series of ethanol, critical-point dried, sputtered with gold, and took photos with SEM.
Statistics were performed using SPSS 21.0 software (IBM). The repeated measures ANOVA were used for comparing live or dead cell counts. Analysis data from multiple groups were carried out by one-way ANOVA followed by Tukey’s multiple comparison tests. Comparison between the two groups was performed by two-tailed Student’s t-test. Friedman test was used for non-parametric test.
Please cite this article as: X. Lei et al., Effects of cyclic fluid stress at different frequencies on behaviors of cells incubated on titanium alloy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.070
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Statistical significance was considered at p < 0.05. 3. Results
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were very similar (Fig. 1B and C) (live cells, F ¼ 0.002, P ¼ 0.968; dead cells, F ¼ 0.171, P ¼ 0.686; n ¼ 9), so did the viabilities of cells (Fig. 1D). The above results supported that Ti-A substrates were cell-friendly and could be used in the following studies.
3.1. Surface characterization The surface images of Ti-A substrates visualized by SEM and AFM were shown in Figs. S2A and B. The data (Fig. S2C) proved that the Ti-A substrates had reached the flat and smooth criterion [6,14]. The microscopies of water droplets statically contacting Ti-A surface were exhibited in Fig. S2D, suggesting that the smooth Ti-A surface was weak hydrophilicity. 3.2. Biocompatibility and cell viability To evaluate the biocompatibility of Ti-A used, NIH-3T3 cells were incubated on Ti-A substrates for 12, 24 and 48 h and the cell viability was evaluated by live/dead assay. The live cells were stained with green color and dead cells with red color. Glass substrates are used as positive controls. Fluorescence images showed that most of the cells on both types of substrates were alive (Fig. 1A). The average number of live/dead cells in the two groups
3.3. Analysis of the fibroblasts morphological responses to fluid shear stress at different frequencies After the NIH-3T3 cells have incubated in dishes for 48 h, we monitored the cell morphological responses to fluid shear stress at different frequencies. The detailed morphology of cells was based on double stained fluorescence images, i.e., F-actin was stained with red and nucleus with blue color. As shown in Fig. 3A and B, the fibroblasts well aligned along the direction of 0.6 Hz shear force, followed by 0.2 and 1.0 Hz groups, while the static control group presented freedom orientation. Similar to the cell angle, the CSI also showed a biphasic trend with the increase of frequency, with peak at the 0.6 Hz group, although the superiority over 1.0 Hz stress was slight (Fig. 2C). SEM images also supported the above results (Fig. S3), suggesting that the above parameters were most sensitive to the 0.6 Hz stress. As for the number of cells, only the 0.6 Hz group had the similar
Fig. 1. Ti-A biocompatibility and cell viability are detected by live/dead assay after NIH-3T3 cells are incubated on Ti-A surfaces for 12, 24 and 48 h. A, The living cells are stained with green, and the dead cells emit red fluorescent. Scale bar, 200 mm. The boxplots reflecting the number of live (B) and dead cells (C) per unit fluorescence field on each substrate, Ti-A or glass (n ¼ 9). D, Quantitative analysis of the proportion of live/dead cells (n ¼ 9). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: X. Lei et al., Effects of cyclic fluid stress at different frequencies on behaviors of cells incubated on titanium alloy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.070
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Fig. 2. Morphological responses of NIH-3T3 cells implanted on Ti-A surfaces to shear stress for 48 h. A, The merged images consist of actin (red) and the DAPI-stained nucleus (blue). The force direction is vertical, except for the static culture group as a negative control. Scale bars: 200 mm. B, The cell orientation in the range of 0e90 is shown with the bar chart. C, Cell shape index (CSI). D, Cell counts. E, Average single cell area. n. s. no statistical significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
cell counts with the static control, which surpassed either 0.2 Hz or 1.0 Hz group (Fig. 2D), which suggested that 0.6 Hz force might increase cell adhesion and proliferation, otherwise, cells were easily washed away from Ti-A substrates by constantly waving fluid. Interestingly, there was no significant difference in cell area between groups but the 1.0 Hz group, which had a maximum value compared with other groups (Fig. 2E).
markers of myofibroblasts, are often used to assess cell differentiation potential [12,13]. The results showed that both of them were most sensitive to 1.0 Hz stress, but no significant difference in other groups (Figs. 3A, C, D, F, G, S5). Thus, 1.0 Hz force promotes fibroblast differentiation into myofibroblasts and lead to the increase of cell area (Fig. 2E), because myofibroblasts had a larger spreading area than fibroblasts [17,18].
3.4. Analysis of fibroblasts adhesion and differentiation reactions to fluid shear stress at different frequencies
3.5. Analysis of BMSCs differentiation reactions to fluid shear stress at 1.0 Hz
To quantitate and compare cell FAs and differentiative potential under fluid stress of different frequency, we selected three protein markers for analysis, including vinculin, Col-I and a-SMA (Figs. 3A, S4, S5). The cell adhesion ability was assessed by fluorescence intensity and mRNA expression of vinculin, which is a force-sensitive protein [15] and is one of the essential components of FA plaques [16]. The image (Fig. S4) and data analyses (Fig. 3B, E) suggested that 0.6 Hz shear force had a more positive impact on cell adhesions, whereas 1.0 Hz stress had a reverse effect, which partially confirmed our hypothesis about the reason for the relatively high number of cells in the 0.6 Hz group (Fig. 2D). We next examined the fluorescence intensity and mRNA expression of Col-I and a-SMA in fibroblasts. Col-I and a-SMA, as
Previous results indicated that 1.0 Hz fluid force contributed to the differentiation of fibroblasts into myofibroblasts, a key event in physiological or pathological tissue healing [7,8]. Therefore, we next used BMSCs to observe the effect of this frequency shear stress on stem cell differentiation, and to understand the specific role of stem cells in skin-implant integration. Immunofluorescence and protein expression results showed that 1.0 Hz stress could increase Col-I content in BMSCs (Fig. 4A, C, E), but did not affect a-SMA expression (Fig. 4A, B, E). Besides, the fluid force also enlarged the cell area compared with the static control (Fig. 4A, D). This suggests that stem cells only participate in collagen regeneration without increasing skin wound contractility, which endowed by aSMA [19].
Please cite this article as: X. Lei et al., Effects of cyclic fluid stress at different frequencies on behaviors of cells incubated on titanium alloy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.070
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Fig. 3. Expression of adhesion and differentiation related genes and fluorescence intensity in fibroblasts for 48h. A, Fluorescence image of a-SMA under high power microscopy. Bar ¼ 20 mm. B-D, Semi-quantitative fluorescence intensity value of vinculin (B), Col-I (C) and a-SMA (D). E-G, the mRNA expression of vinculin (E), Col-I (F) and a-SMA (G). GAPDH served as an internal control (EeG). n. s. no statistical significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
4. Discussion Patients need motion to maintain muscle strength and prevent venous thrombosis after external fixation treatment for limb trauma. Thus, in the design of implant surface modification, it is necessary to consider the effect of movement on sealing of materials. In this work, apart from the intentional 0e1 Hz frequency gradient selection, we also referred to amplitudes of motion of lower limb muscles in a complete gait cycle, and the fascicle length varied with a range of approximately 0.77 cme2.97 cm [20]. Thus, the 2 cm oscillator amplitude we set fit the fascicle length change. Besides, the reciprocating fluid direction provided by our oscillator is also roughly the same as the way the muscles flex and stretch [4,20,21]. Therefore, the shear force model we designed is an excellent way to simulate the mechanical environment around external fixation, regardless of frequency, amplitude and direction. Up to now, the impact of frequency on cell orientation has not yet reached a consensus [22]. For example, at 1.0 Hz, the cell direction was stable at 60e90 under biaxial cyclic deformation [23], but at uniaxial cyclic stretching, the orientation was perpendicular to the direction of the force [24]. For low frequency, 0.1 Hz strain could make the cell direction either perpendicular (for normal skin fibroblasts) or parallel (for C2C12 skeletal muscle cells) to the cyclic strain direction [25,26]. These different conclusions may be attributed to the specificity and uniqueness of the cell type and frequency stress model used in each study. In the reciprocating fluid shear stress model we designed, only 0.6 Hz stress aligned the cell angle with the direction of the force. This discovery implied the presence of a characteristic frequency between 0.6 Hz and 1.0 Hz, and beyond which the cell alignment would regress. The spindle shape of fibroblasts is its intrinsic characteristics [25e27], surprisingly, we found that 0.6 Hz cyclic force enhanced cellular polarization compared to the other groups. Since vinculin is
a key element for sensing extracellular mechanical stimuli and coordinated the force-induced cell polarization [15,16], it is possible that 0.6 Hz increased cell polarity by increasing vincluin expression. Cell polarity is an important indicator of cell migration, a key factor affecting wound healing. The greater the polarity, the faster the cell migration would be [17]. In other words, a good bio-seal requires not only well-aligned fibroblasts to produce parallel and dense ECM around the implant, but also rapid migration to speed up the integration of skin and implants. Our results showed 0.6 Hz stress could achieve the above requirements and improve the sealability of the implant. Differentiation of fibroblasts into myofibroblasts is a key event in physiological wound healing and pathological scar formation [7,8], and is characterized by production of a-SMA and Col-I [12,13]. Here, we found that the expression of a-SMA and Col-I in the 1.0 Hz group were significantly increased compared with the other groups, suggesting that 1.0 Hz shear force could promote fibroblast differentiation, but what we need to pay attention to is that it simultaneously depolymerized FAs. Interestingly, this finding was different from that of the static conditions, in which the size of FAs positively regulated the level of differentiation [7,19]. The reason might be that the high-frequency stress replaced FAs to provide the intracellular tension, an essential factor for activating myofibroblasts, by enhancing the stress fiber stiffness, due to its intrinsic viscosity and no time to relax at high frequency [28]. The myofibroblasts activated by 1.0 Hz stress promoted collagen aggregation and fiber contraction, and increased Col-I produced by stem cells overlapped ECM accumulation. Thus, long-term stimulation of 1.0 Hz shear force may result in fiber encapsulation around the implant, which was not benefit to the improvement of sealing. There are still some limitations in this study. Although the parameters in our self-designed mechanical model are based on human gait cycle, the NIH-3T3 cells and BMSCs used in this study are
Please cite this article as: X. Lei et al., Effects of cyclic fluid stress at different frequencies on behaviors of cells incubated on titanium alloy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.070
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Fig. 4. The differentiation of BMSCs seeded on Ti-A for 48 h. A, Fluorescent images of BMSCs. Images are taken in pseudo-color mode of fluorescence microscopy, including red (Factin), green (a-SMA/Col-I) and blue (nucleus). Scale bars: 100 mm. B, Mean a-SMA fluorescence intensity. C, Mean Col-I fluorescence intensity. D, Mean single cell area. E, Protein expression of a-SMA and Col-I, protein banding (left) and semi-quantitative analysis (middle and right). n. s. no statistical significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
mouse-derived rather than human. In terms of species, mice and humans are close relatives. Several authoritative studies have shown that mouse-derived fibroblasts are highly consistent with human in skin lineage, wound healing and tissue structure with age [29e31]. Treatment of mouse skin ulcers with wound-resident stem cells can provide a new therapeutic approach for human skin wounds [32]. The above literatures show that the research results of mouse-derived cells in the field of skin have great reference and guidance for human beings, of course, not necessarily completely replicated. In addition, the acquisition of mousederived cells is easier than that of humans and does not involve ethical issues. So we used the mouse-derived cells mentioned above to study and conclude that human limb activity may have an impact on skin-implant sealing. Declaration of competing interest The authors have no conflict of interest to declare. Acknowledgements The authors are grateful for the financial supports from the National Key R&D Program of China [No. 2016YFC1100300], the
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Effects of cyclic fluid stress at different frequencies on behaviors of cells incubated on titanium alloy Xing Lei a, b, Hao Wu a, Yue Song a, Bin Liu a, Shuai-Shuai Zhang a, Jun-Qin Li a, Long Bi a, *, Guo-Xian Pei a, ** a b
Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, China Department of Orthopedic Surgery, Linyi People’s Hospital, Linyi, 276000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 October 2019 Accepted 12 November 2019 Available online xxx
The orthopedic external fixation is always in dynamic mechanical environment with the somatic movement. We used a self-designed mini oscillator to simulate this condition by providing the reciprocating cyclic fluid stress, and observed the behavioral responses of fibroblasts implanted on titanium alloy plane to the stress at different frequencies, including 0.2 Hz, 0.6 Hz, and 1.0 Hz. We found that the cell angle, shape index and expression of vinculin were mostly biphasic-dependent with the increase of frequency, with peaks at 0.6 Hz. Whereas the cell area, expression of Col-I and a-SMA were mainly affected by the 1.0 Hz stress. Interestingly, 1.0 Hz stress also promoted Col-I expression of bone marrow mesenchymal stem cells (BMSCs), although it did not increase a-SMA. These results reveal that 0.6 Hz stress improves the alignment, polarity and adherence of fibroblasts on titanium alloy substrates, thus improving the sealing of implants; the 1.0 Hz force activates the differentiation of fibroblasts into myofibroblasts and increases collagen produced by stem cells, which probably cause the formation of fibrous capsules around implants. © 2019 Elsevier Inc. All rights reserved.
Keywords: Cyclic fluid shear stress Fibroblasts BMSCs Alignment Vinculin Differentiation
1. Introduction Many studies have done a lot of work on the surface modification of percutaneous devices to promote the integration of the materials and skin and prevent the pin tract infection [1], but neglected the influence of external mechanical environmental factors on the implant sealing. Especially in orthopedics, implants, such as external fixation, are always in dynamic environment with the somatic movement, which is different from the static stress environment of denture in stomatology. Inspired by the above idea, in this work, we self-designed a mini lab oscillator to provide a reciprocating fluid shear force to simulate the mechanical environment sensed by external fixation, and tried to reveal the behavioral responses of the major participants in skin tissue integration, fibroblasts [2] and stem cells [3], to dynamic mechanical forces. In terms of frequency setting, we took the walking speed of
* Corresponding author. Department of Orthopedics, Xijing hospital, Fourth Military Medical University, 15 changle West street, Xi’an, 710032, China. ** Corresponding author. E-mail addresses: [email protected] (L. Bi), [email protected] (G.-X. Pei).
human lower limbs as a reference. The walking frequency of normal adults is close to 1 Hz (0.97 ± 0.04 Hz) in a gait cycle [4], which is defined from the initial foot contact to the following ipsilateral initial contact. For the elderly or traumatized, the frequency usually becomes smaller (Fig. S1). So in this work, the maximum cycle frequency of the oscillator is set to 1.0 Hz, followed by 0.6 Hz and 0.2 Hz as transitions, with static culture as a control. We observed the morphological responses of fibroblasts planted on titanium alloy plane under fluid stress of different frequency, such as cell angle, polarity, area and number. Theoretically, if the cell alignment is better and the polarity is greater, the extracellular matrix (ECM) produced will be more parallel and ordered, and the sealing of skin-implants will be more good [5]. In addition, we also examined the expression of adhesion and differentiation markers in fibroblasts, including fluorescence intensity, gene or protein expression of vinculin, Col-I and a-SMA, to evaluate the effects of different frequency stresses on the sealing strength of skin-implants [6] and tissue repair ability [7,8]. Because stem cells are involved in the epidermal regeneration and wound healing [9], this study also explored what role stem cells played in skin-implants sealing under dynamic fluid forces by using bone marrow mesenchymal stem cells (BMSCs).
https://doi.org/10.1016/j.bbrc.2019.11.070 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: X. Lei et al., Effects of cyclic fluid stress at different frequencies on behaviors of cells incubated on titanium alloy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.070
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2. Materials and methods
2.6. Immunofluorescence
2.1. Samples preparation and surface characterization
Immunofluorescence was performed by using our previously experimental protocol [11]. The antibodies include: vinculin (Sigma), types I collagen (Col-I) antibody (Abcam),a-SMA (Sigma), goat anti-mouse-FITC (Sigma). F-actin was stained with phalloidinTRITC (Cytoskeleton) and the nucleus with DAPI (Thermo). All specimens were then examined under a fluorescence microscope (Carl Zeiss) at low magnification (5 ) or a laser scanning confocal microscopy (Nikon) at high magnification (40 ). The fluorescent images were quantitatively analyzed using NIH Image J software.
Square TC4 titanium alloy (hereinafter referred to as Ti-A) sheets (10 mm 10 mm length, 1 mm thick) were purchased from Shenzhen Chuangyifu Metal Materials Co., Ltd. The Ti-A substrates were mechanically polished to achieve smooth and mirror like surfaces. All samples were cleaned and sterilized with 90% ethanol for 4 h, and washed with sterile PBS before plating the cells. Surface topography was imaged by LaB6 scanning electron microscopy (SEM) (Tescan) and quantified by atomic force microscopy (AFM) (Agilent). Water contact angle measurements were investigated by JC2000DM contact angle measuring instrument (Zhongyi Kexin Technology Co., Ltd.). 2.2. Cell culture NIH-3T3 (mouse embryonic fibroblast cell line, Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and mouse bone marrow-derived mesenchymal stem cells (BMSCs, which were isolated from the femur bone marrow of 2-week-old mice. See our previous published research for more details [10]) were cultured in DMEM with 10% FBS. NIH-3T3 (2 104/ml) or BMSCs (1 104/ml) were seeded onto the Ti-A or groove substrates and incubated for 8 h in 24-well microplates. Cellular analyses were carried out at 48 h after samples were transferred from microplates to f100mm cell culture dishes. 2.3. Biocompatibility and cell viability assays The biocompatibility of Ti-A sheets was tested by examining the growth of NIH-3T3 cells using the live/dead viability assay. Square glass pieces with the same size as the Ti-A were used as negative control substrates. NIH-3T3 cells were seeded at 1.2 104 cells per sheet. The cells were double stained with Calcein-AM (MedChemExpress) and EthD-1 (Sigma-Aldrich) combined solutions on each substrate for 12, 24 and 48 h. Images were obtained using an Axio Observer A1 optical microscope (Carl Zeiss) equipped with fluorescence light source and filters. The number of live/dead cells were counted and analyzed by using NIH Image J software. 2.4. Construction of fluid shear stress models at different frequencies In order to simulate the mechanical shear stress around the external fixator during muscle flexion and extension in vivo, we designed a mini lab oscillator which can provide reciprocating fluid shear force to act on cells (Video S1). Two f100mm cell culture dishes were placed in parallel on the oscillator, with six Ti-A sheets implanted in each dish. The oscillator can be placed in a cell culture incubator to provide power without affecting the cell culture environment (Video S2). Three grades of 0.2, 0.6 and 1.0 Hz were selected as the experimental groups, and the amplitude is 2 cm; cells cultured in static state were used as a control. The oscillator worked 18 h/day.
2.7. Morphological analysis At low magnification, nine visual fields per sample were randomly captured for morphological analysis. Cell orientation was defined as the angle between the long axis of cells and the direction of the shear force; cell shape index (CSI) was based on the ratio of the long axis to the short axis. The inclusion criteria of the above two indicators: (i) Cell outline is clear; (ii) Showing cell polarity. Exclusion criteria: (i) intercellular fusion leads to unclear contour; (ii) more than 2 branches; (iii) obviously curved cells; (iv) binuclear or multinucleated cells. Besides, because of the very small proportions in each group, mononuclear spherical cells were not calculated, either. The number of cells was quantified by counting nuclei. The cell area and the fluorescence intensity of F-actin were also measured in this work. At high magnification, more than five images per specimen were randomly visualized for morphological analysis. FAs were quantified using an anti-vinculin antibody, including fluorescence intensity, FA area and number per cell, and FA area per focal contact point. Differentiation from fibroblasts to myofibroblasts was determined by analyzing fluorescence intensity of Col-I and a-SMA [12,13]. All images were analyzed with NIH Image J software. 2.8. RNA purification, semi-quantitative RT-PCR (sqPCR) and quantitative RT-PCR (qPCR) qPCR was performed using standard protocol as our previously described [11]. Total RNA from the treated cells was extracted using the E. Z.N.A.® Total RNA Kit I (Omega). Total RNA was quantified with a spectrophotometer. Reverse transcription was performed using the two-step PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). sqPCR was performed using Taq™ PCR mix (Takara). The 2 DDCt method was used for data analysis, and mRNA expression was shown relative to control 24 h group. The primers, designed by Takara Biotechnology Co., Ltd., are shown in Table S1. 2.9. Western blotting WB analysis was performed using a standardized protocol as described previously [10]. The antibodies include: Col-I antibody (Abcam), a-SMA (Sigma), b-actin (Affinity), and HRP-linked goat anti-mouse IgG (H þ L) (Affinity). 2.10. Statistical analysis
2.5. SEM After 48 h of fluid force acting on NIH-3T3 cells seeded onto the Ti-A substrates, cell morphologies were assessed via SEM (Tescan). Briefly, the samples were fixed with 2.5% glutaraldehyde at 4 C for 4 h, dehydrated through a graded series of ethanol, critical-point dried, sputtered with gold, and took photos with SEM.
Statistics were performed using SPSS 21.0 software (IBM). The repeated measures ANOVA were used for comparing live or dead cell counts. Analysis data from multiple groups were carried out by one-way ANOVA followed by Tukey’s multiple comparison tests. Comparison between the two groups was performed by two-tailed Student’s t-test. Friedman test was used for non-parametric test.
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Statistical significance was considered at p < 0.05. 3. Results
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were very similar (Fig. 1B and C) (live cells, F ¼ 0.002, P ¼ 0.968; dead cells, F ¼ 0.171, P ¼ 0.686; n ¼ 9), so did the viabilities of cells (Fig. 1D). The above results supported that Ti-A substrates were cell-friendly and could be used in the following studies.
3.1. Surface characterization The surface images of Ti-A substrates visualized by SEM and AFM were shown in Figs. S2A and B. The data (Fig. S2C) proved that the Ti-A substrates had reached the flat and smooth criterion [6,14]. The microscopies of water droplets statically contacting Ti-A surface were exhibited in Fig. S2D, suggesting that the smooth Ti-A surface was weak hydrophilicity. 3.2. Biocompatibility and cell viability To evaluate the biocompatibility of Ti-A used, NIH-3T3 cells were incubated on Ti-A substrates for 12, 24 and 48 h and the cell viability was evaluated by live/dead assay. The live cells were stained with green color and dead cells with red color. Glass substrates are used as positive controls. Fluorescence images showed that most of the cells on both types of substrates were alive (Fig. 1A). The average number of live/dead cells in the two groups
3.3. Analysis of the fibroblasts morphological responses to fluid shear stress at different frequencies After the NIH-3T3 cells have incubated in dishes for 48 h, we monitored the cell morphological responses to fluid shear stress at different frequencies. The detailed morphology of cells was based on double stained fluorescence images, i.e., F-actin was stained with red and nucleus with blue color. As shown in Fig. 3A and B, the fibroblasts well aligned along the direction of 0.6 Hz shear force, followed by 0.2 and 1.0 Hz groups, while the static control group presented freedom orientation. Similar to the cell angle, the CSI also showed a biphasic trend with the increase of frequency, with peak at the 0.6 Hz group, although the superiority over 1.0 Hz stress was slight (Fig. 2C). SEM images also supported the above results (Fig. S3), suggesting that the above parameters were most sensitive to the 0.6 Hz stress. As for the number of cells, only the 0.6 Hz group had the similar
Fig. 1. Ti-A biocompatibility and cell viability are detected by live/dead assay after NIH-3T3 cells are incubated on Ti-A surfaces for 12, 24 and 48 h. A, The living cells are stained with green, and the dead cells emit red fluorescent. Scale bar, 200 mm. The boxplots reflecting the number of live (B) and dead cells (C) per unit fluorescence field on each substrate, Ti-A or glass (n ¼ 9). D, Quantitative analysis of the proportion of live/dead cells (n ¼ 9). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 2. Morphological responses of NIH-3T3 cells implanted on Ti-A surfaces to shear stress for 48 h. A, The merged images consist of actin (red) and the DAPI-stained nucleus (blue). The force direction is vertical, except for the static culture group as a negative control. Scale bars: 200 mm. B, The cell orientation in the range of 0e90 is shown with the bar chart. C, Cell shape index (CSI). D, Cell counts. E, Average single cell area. n. s. no statistical significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
cell counts with the static control, which surpassed either 0.2 Hz or 1.0 Hz group (Fig. 2D), which suggested that 0.6 Hz force might increase cell adhesion and proliferation, otherwise, cells were easily washed away from Ti-A substrates by constantly waving fluid. Interestingly, there was no significant difference in cell area between groups but the 1.0 Hz group, which had a maximum value compared with other groups (Fig. 2E).
markers of myofibroblasts, are often used to assess cell differentiation potential [12,13]. The results showed that both of them were most sensitive to 1.0 Hz stress, but no significant difference in other groups (Figs. 3A, C, D, F, G, S5). Thus, 1.0 Hz force promotes fibroblast differentiation into myofibroblasts and lead to the increase of cell area (Fig. 2E), because myofibroblasts had a larger spreading area than fibroblasts [17,18].
3.4. Analysis of fibroblasts adhesion and differentiation reactions to fluid shear stress at different frequencies
3.5. Analysis of BMSCs differentiation reactions to fluid shear stress at 1.0 Hz
To quantitate and compare cell FAs and differentiative potential under fluid stress of different frequency, we selected three protein markers for analysis, including vinculin, Col-I and a-SMA (Figs. 3A, S4, S5). The cell adhesion ability was assessed by fluorescence intensity and mRNA expression of vinculin, which is a force-sensitive protein [15] and is one of the essential components of FA plaques [16]. The image (Fig. S4) and data analyses (Fig. 3B, E) suggested that 0.6 Hz shear force had a more positive impact on cell adhesions, whereas 1.0 Hz stress had a reverse effect, which partially confirmed our hypothesis about the reason for the relatively high number of cells in the 0.6 Hz group (Fig. 2D). We next examined the fluorescence intensity and mRNA expression of Col-I and a-SMA in fibroblasts. Col-I and a-SMA, as
Previous results indicated that 1.0 Hz fluid force contributed to the differentiation of fibroblasts into myofibroblasts, a key event in physiological or pathological tissue healing [7,8]. Therefore, we next used BMSCs to observe the effect of this frequency shear stress on stem cell differentiation, and to understand the specific role of stem cells in skin-implant integration. Immunofluorescence and protein expression results showed that 1.0 Hz stress could increase Col-I content in BMSCs (Fig. 4A, C, E), but did not affect a-SMA expression (Fig. 4A, B, E). Besides, the fluid force also enlarged the cell area compared with the static control (Fig. 4A, D). This suggests that stem cells only participate in collagen regeneration without increasing skin wound contractility, which endowed by aSMA [19].
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Fig. 3. Expression of adhesion and differentiation related genes and fluorescence intensity in fibroblasts for 48h. A, Fluorescence image of a-SMA under high power microscopy. Bar ¼ 20 mm. B-D, Semi-quantitative fluorescence intensity value of vinculin (B), Col-I (C) and a-SMA (D). E-G, the mRNA expression of vinculin (E), Col-I (F) and a-SMA (G). GAPDH served as an internal control (EeG). n. s. no statistical significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
4. Discussion Patients need motion to maintain muscle strength and prevent venous thrombosis after external fixation treatment for limb trauma. Thus, in the design of implant surface modification, it is necessary to consider the effect of movement on sealing of materials. In this work, apart from the intentional 0e1 Hz frequency gradient selection, we also referred to amplitudes of motion of lower limb muscles in a complete gait cycle, and the fascicle length varied with a range of approximately 0.77 cme2.97 cm [20]. Thus, the 2 cm oscillator amplitude we set fit the fascicle length change. Besides, the reciprocating fluid direction provided by our oscillator is also roughly the same as the way the muscles flex and stretch [4,20,21]. Therefore, the shear force model we designed is an excellent way to simulate the mechanical environment around external fixation, regardless of frequency, amplitude and direction. Up to now, the impact of frequency on cell orientation has not yet reached a consensus [22]. For example, at 1.0 Hz, the cell direction was stable at 60e90 under biaxial cyclic deformation [23], but at uniaxial cyclic stretching, the orientation was perpendicular to the direction of the force [24]. For low frequency, 0.1 Hz strain could make the cell direction either perpendicular (for normal skin fibroblasts) or parallel (for C2C12 skeletal muscle cells) to the cyclic strain direction [25,26]. These different conclusions may be attributed to the specificity and uniqueness of the cell type and frequency stress model used in each study. In the reciprocating fluid shear stress model we designed, only 0.6 Hz stress aligned the cell angle with the direction of the force. This discovery implied the presence of a characteristic frequency between 0.6 Hz and 1.0 Hz, and beyond which the cell alignment would regress. The spindle shape of fibroblasts is its intrinsic characteristics [25e27], surprisingly, we found that 0.6 Hz cyclic force enhanced cellular polarization compared to the other groups. Since vinculin is
a key element for sensing extracellular mechanical stimuli and coordinated the force-induced cell polarization [15,16], it is possible that 0.6 Hz increased cell polarity by increasing vincluin expression. Cell polarity is an important indicator of cell migration, a key factor affecting wound healing. The greater the polarity, the faster the cell migration would be [17]. In other words, a good bio-seal requires not only well-aligned fibroblasts to produce parallel and dense ECM around the implant, but also rapid migration to speed up the integration of skin and implants. Our results showed 0.6 Hz stress could achieve the above requirements and improve the sealability of the implant. Differentiation of fibroblasts into myofibroblasts is a key event in physiological wound healing and pathological scar formation [7,8], and is characterized by production of a-SMA and Col-I [12,13]. Here, we found that the expression of a-SMA and Col-I in the 1.0 Hz group were significantly increased compared with the other groups, suggesting that 1.0 Hz shear force could promote fibroblast differentiation, but what we need to pay attention to is that it simultaneously depolymerized FAs. Interestingly, this finding was different from that of the static conditions, in which the size of FAs positively regulated the level of differentiation [7,19]. The reason might be that the high-frequency stress replaced FAs to provide the intracellular tension, an essential factor for activating myofibroblasts, by enhancing the stress fiber stiffness, due to its intrinsic viscosity and no time to relax at high frequency [28]. The myofibroblasts activated by 1.0 Hz stress promoted collagen aggregation and fiber contraction, and increased Col-I produced by stem cells overlapped ECM accumulation. Thus, long-term stimulation of 1.0 Hz shear force may result in fiber encapsulation around the implant, which was not benefit to the improvement of sealing. There are still some limitations in this study. Although the parameters in our self-designed mechanical model are based on human gait cycle, the NIH-3T3 cells and BMSCs used in this study are
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Fig. 4. The differentiation of BMSCs seeded on Ti-A for 48 h. A, Fluorescent images of BMSCs. Images are taken in pseudo-color mode of fluorescence microscopy, including red (Factin), green (a-SMA/Col-I) and blue (nucleus). Scale bars: 100 mm. B, Mean a-SMA fluorescence intensity. C, Mean Col-I fluorescence intensity. D, Mean single cell area. E, Protein expression of a-SMA and Col-I, protein banding (left) and semi-quantitative analysis (middle and right). n. s. no statistical significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
mouse-derived rather than human. In terms of species, mice and humans are close relatives. Several authoritative studies have shown that mouse-derived fibroblasts are highly consistent with human in skin lineage, wound healing and tissue structure with age [29e31]. Treatment of mouse skin ulcers with wound-resident stem cells can provide a new therapeutic approach for human skin wounds [32]. The above literatures show that the research results of mouse-derived cells in the field of skin have great reference and guidance for human beings, of course, not necessarily completely replicated. In addition, the acquisition of mousederived cells is easier than that of humans and does not involve ethical issues. So we used the mouse-derived cells mentioned above to study and conclude that human limb activity may have an impact on skin-implant sealing. Declaration of competing interest The authors have no conflict of interest to declare. Acknowledgements The authors are grateful for the financial supports from the National Key R&D Program of China [No. 2016YFC1100300], the
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Please cite this article as: X. Lei et al., Effects of cyclic fluid stress at different frequencies on behaviors of cells incubated on titanium alloy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.070