Responses of osteoblasts under varied tensile stress types induced by stretching basement materials

ABSTRACT

Osteoblasts are mechanosensitive cells. Tensile stress with different conditions, including loading time, frequency, magnitude, etc. would cause varied responses in osteoblasts. However, it was not clarified that the effect of the loading types on the osteoblasts. In this study, we focused on the effect of varied tensile stress types on osteoblasts, including isotropic stretch, biaxial stretch, and uniaxial stretch with the negative ratio of transverse strain to axial strain (NR) −1, 0, and 0.2 respectively. Cell proliferation was determined to be most efficient when stimulated by 6% strain at a frequency of 1 Hz and a negative value of 0 for 1 h/day. The varied strain resulted in a thickening of the F-actin cytoskeleton and a thinning of the nucleus. Nuclear flattening caused Yes-associated protein (YAP) to be transported to the nucleus. It was suggested that the influence of loading types on the mechanobiology responses must be noticed. The mechanism of cell mechanical sensitivity under varied loading types was explored, which would provide good suggestions for designing microstructures to control deformation patterns in bone tissue engineering.

GRAPHICAL ABSTRACT

KEYWORDS:

• MC3T3-E1
• tensile stress
• negative poisson’s ratio
• cell proliferation
• YAP

Introduction

The growth, absorption, and reconstruction of bone are related to the mechanical environment [1]. Osteoblasts, osteoclasts, and osteocytes respond to complex mechanical stimuli in vivo like shear, tensile, and compressive forces [2,3]. They cooperate to maintain the normal functions of bone [4]. Osteoblasts, located on the bone surface, can form suitable bone by sensing the strain of the bone caused by mechanical stress [5]. The mechanical sensitivity of osteoblasts has been studied in detail for decades. It has been found that stretching osteoblasts can regulate cell proliferation [6], differentiation [7,8], apoptosis [9], and change the expression of collagen fibers and their arrangement in the extracellular space [5]. The current research mainly focused on the strain, loading time, and frequency, ignoring the influence of the loading type.

Different loading types were used in previous studies, it is impossible to uniformly compare and discuss the results when there is a discrepancy in experimental conclusions. The tensile stress generated by the four-point bending method was used to stretch MC3T3-E1 cells, and it was found that under the loading condition of 0.5 Hz and 1 h/Day, the cell proliferation was promoted at 2.5% strain and inhibited at 5% strain compared with the static group [6]. In another study, uniaxial stretch with 10% strain was applied to osteoblasts that grew on a piece of rectangular rubber membrane with a circular defect in the middle at a frequency of 0.2 Hz for 6 h, and it was found that strain applied to cells promoted cell proliferation compared to the static group [10]. For different loading methods, there is a great difference in the strain size that promotes cell proliferation. isotropic tensile strains of 0.8%, 1.6%, 2.4%, and 3.2% respectively for 48 h at a frequency of 1 Hz were loaded to human osteoblasts in Flexcell-5000TM stretch devices. Compared with the static control group, the alkaline phosphatase (ALP) activity was significantly increased when the strain was 0.8% [11]. Uniaxial tensile stress with 1% strain was applied to human osteoblasts for 2 days, 30 mins per day at a frequency of 1 Hz and the ALP activity of the cells was significantly reduced [12]. The two groups of experiments mentioned above produced different experimental results because of different experimental conditions. Besides the varied strain, time, and frequency, the loading methods were also different. Different loading methods will produce different tensile stresses on cells in different directions, which would greatly affect the cell’s feedback to mechanical stimulation.

Different loading types can be used to simulate different Poisson’s ratios. Poisson’s ratio was used to describe the relationship between transverse and longitudinal strains of a material under stress and deformation. Poisson’s ratio is the negative ratio of transverse strain to longitudinal strain when the material is subjected to uniaxial stress. Most materials have positive Poisson’s ratio, that is, when uniaxial compression (tension) during plastic deformation, they expand (contract) in the direction orthogonal to the applied load, while negative Poisson’s ratio materials, in contrast, contract (expand) in the direction orthogonal to the applied load during uniaxial compression (tension).

The mechanical properties and preparation of structures with negative Poisson’s ratio characteristics in implants have been explored [13]. The structures have also been found to play an interesting role in many ways. For example, under the stimulation of periodic tensile stress, the degradation rate of the scaffolds with negative Poisson ratio is higher than that of the scaffolds with positive Poisson ratio [14]. Compared to the porous plate with a positive Poisson’s ratio, the porous plate with a negative Poisson’s ratio had the advantages of delayed crack initiation and more than 20% longer fatigue life [15]. Porous elements with auxetic properties have also been found in walnut shells to decrease their fracture behavior [16]. The unique deformation characteristics of the negative Poisson’s ratio structure also contributed to bone nail fixation, which is expected to solve the current loosening problem through the structure without relying on fixed components that can create stress concentration [17–20]. In addition, the deformation of structures with a negative Poisson’s ratio can also affect the proliferation and differentiation of stem cells [21,22]. Polylactic-co-glycolic acid (PLGA) scaffolds with different Poisson’s ratios cultured with osteoblasts under pressure. The results showed that the scaffold with the negative Poisson’s ratio promoted the proliferation of osteoblasts, the dynamic loading further deepened this effect [23,24]. However, the biological effects and mechanisms of different Poisson’s ratio stretching on osteoblasts need to be further explored. Different loading types can be used to simulate different Poisson’s ratios to explore the effects Poisson’s ratios on osteoblasts.

In this study, we modified the Flexcell-5000TM stretch device to achieve different loading types with the negative ratio of transverse strain to axial strain (NR) −1, 0, and 0.2 respectively, and explored the effects of different loading types on osteoblast proliferation and the mechanisms. It would be helpful to further understand the response and mechanism of osteoblasts under complex mechanical environments and can guide the application of different Poisson’s ratio structures in bone implants.

Methods

Cell culture

The preosteoblast MC3T3-E1 Subclone 14 cell line was purchased from the American type culture collection (ATCC). Cells were cultured in Alpha Modified Minimum Essential Medium (α-MEM) (Genview, GM3127) supplemented with 10% fetal bovine serum (FBS) (Every Green 11,011–8611) and 1% penicillin and streptomycin (Gibco,14150122). Cells were cultured at a density of 2 × 10⁴/cm². On reaching 80–90% confluence, the cells were digested with trypsin-0.53 mM EDTA (Gibco 25,200,056), and centrifuged for subsequent experiments.

Customization of loading types

The principle of the Flexcell-5000TM stretch device was shown in Figure 1(a). The cells, growing on the rubber membrane of the BioFlex culture plate (BF3001U), were stretched by vacuuming the space between the gasket and loading post. Therefore, by customizing three types of polytetrafluoroethylene loading posts shown in Figure 1(b) to adjust the space between the gasket and loading post, the Flexcell-5000TM stretch device could generate different sizes of stretch strain in different directions on the rubber membrane of the BioFlex culture plate. The strain was measured by Digital Image Correlation (DIC) equipment and analyzed by Matlab software to produce a strain distribution diagram shown in Figure 2(a). We achieved three loading types with the negative ratio of transverse strain (Y) to axial strain(X) −1, 0, and 0.2 respectively.

Figure 1. Establishment of different loading types. (a) Flexcell-5000TM device stretching principle diagram. The rubber membrane inoculated with cells is stretched by extracting the air between the gasket and the loading base. (b) Different shapes of loading posts and their corresponding stretching patterns. By changing the shape of the loading post, the stretching of the rubber membrane is adjusted. (c) Strain distribution of the rubber membrane in X and Y directions.

Figure 2. (a) The process of measuring strain by Digital Image Correlation (DIC) equipment. The strain size is obtained by Matlab software to obtain the range of uniform strain distribution. (b) Control cell growth in the range of uniform strain. The cells were cultured on a BioFlex culture plate for 7 days and basically grew in the range of uniform strain. Scale bar: 500 μm.

Adjusting the distribution area where the cells grow on the rubber membrane

The experiment took advantage of the characteristic that the rubber membrane only incubated with fibronectin (FN) on the surface could make cells grow. The portion distributed uniformly strain, according to DIC detection results, was removed from a piece of circular silicone film which owns the same aperture size as the BioFlex culture plate. The cut silicone film was cleaned and soaked in 75% alcohol for 1 hour to sterilize, then dried and affixed to the BioFlex culture plate. 1 mL FN solution (Solarbio, F8180), diluted with sterile phosphate-buffered saline (PBS) (Solarbio, P1020) to 10 $\mathit{\mu }$g/mL, was added to each well for 45 mins. 1 mL cell suspension with a density of 5000/cm² was added to each well for uniform inoculation. After 3 h of inoculation, the cut silicone film was removed. Then, 2 mL media were added to each well. The BioFlex culture plates were cultured in the incubator.

Conditions and applications of cyclic stretch

Cyclic stretch was applied to cells at a frequency of 1 Hz for 1 h/day on the day after inoculation. The experiment was divided into two groups. Experimental group 1 (EG1) applied strains of 3%, 6%, and 9% to cells when NR was 0. Experimental group 2 (EG2) applied strains of −1, 0, 0.2 NR with the controlled strain which is according to the results of EG1. Both EG1 and EG2 contain three different loading conditions. Cells without mechanical stretch were used as a control group.

Cell proliferation

The number of viable cells was indirectly quantified using a cell counting kit-8 (CCK-8) (Biorigin, BN15201) after 7 days. 1 mL of 10% (v/v) CCK-8 reagent was added to each well and incubated in the cell incubator for 3 h. 200 $\mathit{\mu }$L of the reactant was moved intoL of the reactant was moved into a 96-well plate. The absorbance was measured at 450 nm using a multiscan spectrometer (Varioskan Flash; Thermo Electron Corporation). The assays were performed with three biological replicates and there were six samples per replicate.

Mechanical testing of rubber membranes

The rubber membranes of the BioFlex culture plates were cultured with MC3T3-E1 cells and stretched with the conditions of cyclic stretch mentioned above in EG1 and EG2 for 7 days. The rubber membranes were cut into tensile specimens conforming to ISO-527-5B, and the thicknesses of the membranes were measured with the micrometer. Five specimens for each group were tested to generate average data. The mechanical tensile tests of the samples were carried out by the in-situ biaxial fatigue test system. In the mechanical tensile testes, the samples were stretched at a speed of 1 mm/min. The elastic modulus of the membranes was calculated. There were 6 samples for each group.

Immunocytochemistry

After 1 day of inoculation, the cells were stretched at a frequency of 1 Hz for 1 h/day. The media was removed after stretching. The cells were fixed with 4% paraformaldehyde (Solarbio, P1110) and permeabilized with 0.2% Triton X-100 (Solarbio, T8200) for intracellular markers, followed by blocking and incubating with Alexa Fluor®594 Phalloidin (Invitrogen, A12379). After washing, the cells were incubated with rabbit-derived anti-YAP (Abcam, ab205270) and then with Alexa Fluor™ Plus 647 (Invitrogen A32733). The samples were sealed with a DAPI-containing fluoro-shield (Sigma, F6057) and visualized using a confocal microscope (Andor Dragonfly) with a layer scan thickness of 0.5 μm. Nuclear models were obtained by Imaris software. Parameters like the nuclear thickness, volume, and surface area were measured. The stacks of images were projected along the Z-axis to one image with max intensity by ImageJ software. Parameters such as cytoskeletal thickness shown by the white arrows in Figure 4(a), cell area, nucleus area, and the expression of YAP were measured. Three biological replicates were performed and totally 180 cells were measured.

Cell arrangement

The cells were subjected to cyclic stretch with a frequency of 1 Hz and NR of −1, 0, 0.2. Take photos at 1, 3, 5, and 7 h. The effect of stretch with different NR on cell arrangement was observed.

Statistics

Analysis of unpaired sample t-tests was performed by GraphPad Prism 9 to determine significance. Significance levels were set at *p < 0.05, **p < 0.01, and***p < 0.001.

Results

Strains and poisson’s ratios of different loading types

As shown in Figure 1(c), the uniform distribution ranges and the strains of the different loading types produced by the three types of loading posts were shown on the strain distribution diagrams. The average strain values of strain (εX), lateral strain (εY), and NR are shown in Table 1. The actual images and videos of the stretching membrane were shown in the supplementary material.

Table 1. Average transverse (X) and longitudinal (Y) strain values.

Loading post	Type1		Type2		Type3
Direction	X	Y	X	Y	X	Y
3%	3.2%	3.2%	3.0%	0.1%	3.2%	−0.59%
6%	5.8%	5.8%	6.1%	0.4%	6.6%	−1.5%
9%	8.8%	8.6%	8.9%	0.3%	9.4%	−2.0%
Negative Ratio	−1		0		0.2

Cell proliferation

As shown in Figure 2(b), the cells grew at the site with uniform strain 7 days after inoculation. In EG1, MC3T3-E1 cells underwent three groups of different sizes of strain, {εx3%, εy0%}, {εx6%, εy0%}, {εx9%, εy0%} respectively, named X3Y0, X6Y0, X9Y0, the unstressed group serving as the control group (X0Y0). Higher CCK-8 activity was observed over 7 days for the X6Y0 and X0Y0 groups compared with the others (see Figure 3(a)). The EG2 experiment was conducted according to the results of EG1, with εX controlled at 6% and NR changed to −1, 0, and 0.2, the corresponding strains were {εx6%, εy6%}, {εx6%, εy0%}, {εx6%, εy-1.5%}, named X6Y6, X6Y0, X6Y- respectively, with the unstressed group serving as the control group (X0Y0). Higher CCK-8 activity was observed over 7 days in the groups X6Y0 and X0Y0 compared with the others (see Figure 3(b)).

Figure 3. (a, b) CCK-8 assay results in cell proliferation at 7 Days. (c, d) Elastic modulus of rubber membranes from BioFlex culture plate after cultured with MC3T3-E1 cells which were stretched for 1 h/day for 7 days. Data are expressed as mean ± SD, (n = 6). * p < 0.05, ** p < 0.01.

Elastic modulus of rubber membrane under different loading types

As shown in Figure 3(c,d), the elastic moduli of the stretched rubber membranes were significantly lower than that of the without stretching. The elastic moduli of the rubber membranes in the X9Y0 and X6Y- groups were slightly lower than those in the other strain-applied groups.

The F-actin cytoskeleton’s size and the cell/nucleus’ area

In EG1, the F-actin cytoskeleton of cells in the X9Y0 group became thinner and no significant difference was shown between group X3Y0 and X6Y0 (see Figure 4(a,b)). In EG2, the F-actin cytoskeleton was thicker in group X6Y0, and there was no significant difference between group X6Y6 and X6Y- (see Figure 4(f,g)). In both EG1 and EG2, the nuclear area and cell area of the group X6Y0 increased (see Figure 4(c,d,h,i)）, but the proportion of nuclear area in the overall area of the cell decreased (see Figure 4(e,j)).

Figure 4. Immunofluorescence imaging, F-actin cytoskeleton’s size, and the cell/nucleus area. (a, f) Immunofluorescence imaging of MC3T3-E1 cells cultured under different stretch conditions. Scale bar: 50 μm for Nuclei, F-actin, and Merge, 10 μm for the close-up view; (b, g) Cytoskeletal thickness; (c, d, h, i) the area of the nucleus and cytoplasm; (e, j) Area ratio of nucleus to cytoplasm. Data are expressed as violin plot (For nuclear area, cell area, and nuclear area ratio: n = 180. For cytoskeletal thickness: a total of 180 cells, each actin filament overlapped with the nucleus was measured). * p < 0.05, ** p < 0.01, *** p < 0.001.

Thickness, volume, and surface area of the nucleus

As shown in Figure 5(a,e), three-dimensional nuclear models were obtained by Imaris software. For EG1 and EG2, the cell nucleus thickness of group X6Y0 was reduced (see Figure 5(b,f)). In EG1, the nuclear volume of group X3Y0 was smaller than those of group X6Y0 and X9Y0 (see Figure 5(c)). There was no significant difference between the nuclear volume of group X6Y0 and X9Y0 (see Figure 5(c)). In EG2, the nuclear volume of group X6Y6 and X6Y0 showed no significant difference and the volume of group X6Y- was smaller than those (see Figure 5(g)). In both EG1 and EG2, the nuclear surface area of the X6Y0 group was larger than those of the other dynamic groups (see Figure 5(d,h)).

Figure 5. Three-dimensional nuclear models and related parameters (a, e) Three-dimensional nuclear model of MC3T3-E1 cells cultured under different stretch conditions, Scale bar: 10 $\mathit{\mu }$m; (b, f) Nuclear thickness; (c, g) Nuclear volume; (d, h) Surface area of the nucleus. Data are expressed as violin plots (n = 180). * p < 0.05, ** p < 0.01, *** p < 0.001.

Expression of YAP in the nucleus and cytoplasm

As shown in Figure 6(a,e）), YAP and nucleus staining were performed on stretched cells in each group. In EG1, the total and average expression of YAP in the nucleus of group X6Y0 were the highest (see Figure 6(b,c). The average intensity of YAP in the cytoplasm of group X3Y0 was lower than that in group X9Y0 (see Figure 6(d)). In EG2, the overall YAP expression in the nucleus of group X6Y0 was the highest, while that in group X6Y- was significantly lower (see Figure 6(f)). The average fluorescence intensity of YAP in the nucleus of group X6Y0 was the highest, but there was no significant difference among the three groups (see Figure 6(g)). The average cytoplasmic YAP expression in group X6Y0 was decreased (see Figure 6(h)).

Figure 6. Expression of YAP. (a, e) Fluorescent images of YAP (red) and nuclei (blue), Scale bar: 50 μm for Nuclei, YAP and Merge; (b, f) Quantification of total YAP expression of nucleus for MC3T3-E1 cells; (c, g) Quantification of mean YAP expression of nucleus for MC3T3-E1 cells; (d, h) Quantification of mean YAP expression of cytoplasm for MC3T3-E1 cells. Data were expressed as violin plots. *p < 0.05, **p < 0.01, ***p < 0.001.

The arrangement of cells after stretching with different poisson’s ratios

The arrangement of cells at different time points of sustained stretch is shown in Figure 7. The blue arrows represent the direction in which the cells are stressed. There was no significant change in cell arrangement at 1 h for the three groups. At all subsequent time points, the cell arrangement in the X6Y6 group showed no obvious signs of being affected by stretch. At 3 h, the cells in the X6Y0 group were clearly aligned perpendicular to the stretch direction, and the cells in the X6Y- group also tended to line up perpendicular to the stretch direction. At the time points of 5 h and 7 h, the parallelism of cell arrangement in the X6Y0 group and X6Y- group gradually became more obvious, but the degree of parallelism in the X6Y- group was less than that in the X6Y0 group.

Figure 7. Cell arrangement at different time points undergoing strains with different Poisson’s ratios. Scale bar: 500 μm.

Discussion

Osteoblasts are mechanically sensitive cells and are responsible for bone formation. Many studies paid attention to the influence of the strain, time, and frequency of tensile stress on osteoblasts, ignoring the different tensile stresses on cells in different directions generated by different loading types, which also has a great impact on the biomechanical feedback of osteoblasts. It is significant to understand the feedback and mechanism of cell response to different loading types. In this study, we focused on the effects and mechanisms of different strain sizes and loading types, including isotropic, biaxial, and uniaxial stretches, on osteoblast proliferation. It was found that cell proliferation was determined to be most efficient when stimulated by mechanical strain at a frequency of 1 Hz, intensities of 6%, and NR of 0 with a periodicity of 1 h/day. The applied mechanical strain resulted in a thickening of the F-actin cytoskeleton and a thinning of the nucleus. Nuclear flattening caused YAP to be transported to the nucleus. In this experiment, there are a lot of interesting data worthy of discussion.

Effect of rubber membrane’s elastic modulus on cell proliferation

In EG1, group X3Y0 and group X9Y0 inhibited proliferation compared with the static control group X0Y0. In EG2, group X6Y6 and group X6Y- also inhibited proliferation compared with the static control group X0Y0. According to previous studies, compared with static control, proliferation will be promoted when frequency, size, time and other conditions of dynamic strain are not large enough, which will be inhibited after conditions reach the peak value [25]. It was found that when the tensile stress was generated by the four-point bending method, the proliferation of MC3T3-E1 cells was promoted when the strain was 2.5% for 1 h and inhibited when the strain was 5% [6]. According to previous studies, the inhibition of proliferation in X9Y0 and X6Y6 groups may be due to excessive strain. However, when group X6Y0 did not inhibit proliferation, it should have promoted cell proliferation for group X3Y0.

It was found in the experiment that the rubber membrane of the BioFlex culture plate showed relaxation and deformation after stretching. Therefore, mechanical tests were conducted on the stretched rubber membrane. The elastic modulus of the stretched rubber membrane decreased significantly after stretching. Cells can recognize the mechanical properties of the matrix and respond accordingly. One study showed that cell proliferation was promoted when the elastic modulus of hydrogels increased in three hydrogels (11.78 kPa, 21.6 kPa, 38.98 kPa) [26]. The tensile modulus (5.98 ± 0.61 MPa, 7.84 ± 0.13 MPa, 12.4 ± 0.1 MPa) was increased by adjusting HA components (10%, 20%, and 30%) in PCLDA/HA composites. With the increase of tensile modulus, the adhesion, proliferation, and differentiation of MC3T3-E1 cells were enhanced [27]. Cells are mechanically sensitive and can respond to microenvironmental mechanical cues. Cells prefer an external environment with mechanical properties similar to those of the original tissue. For example, in soft tissues such as the vessels and muscles, hydrogel is a better choice [28–30]. For osteoblasts, the enhancement of the mechanical properties of the base is more conducive to the proliferation and differentiation of osteoblasts. So it can be inferred that because the elastic modulus of the rubber membrane decreased after stretching, the cell proliferation in the dynamic group was inferior to that in the static group during long-term culture. Therefore, in similar experiments, we should think about whether the mechanical properties of the substrate can be maintained by mechanical stimulation. The stretch condition and the mechanical properties of the rubber membrane in the X0Y0 group were both different and which not meet the condition of a single control variable. Therefore, the X0Y0 group was not considered in the subsequent experiment.

Effect of tensile stress with different loading types on cell mechanical conduction

External physical forces can be transferred into the nucleus through components such as integrin, actin, and nuclear skeleton to change gene expression [31]. Actin stress fibers and actomyosin contractility can exert vertical and lateral inward compressive forces on the nucleus. Lateral actin fibers can lead to nuclear deformation when cells migrate or are stretched [32,33]. The vertical compression force is applied by the apical actin stress fibers, which form a dome-like structure across the nucleus and are physically attached to the nuclear layer by the LINC (linker of nucleoskeleton and cytoskeleton) complex [34]. On a flat rigid matrix, these forces flatten the nucleus during cell diffusion and can lead to a nuclear film rupture event [35–37].

In the X6Y0 group, the actin stress fiber thickness was increased, resulting in greater pressure on the nucleus. A study has shown that cells can generate greater internal forces when the area is enlarged [38]. The area of cells in the X6Y0 group was larger than others, so it is presumed that greater internal forces were generated in the cells in the X6Y0 group. The trend of cell and nuclear area expansion is similar, the larger the cell area, the larger the nuclear area. However, the expansion is not proportional, with the increase of cell area, the ratio of nucleus to cell area is reduced. In EG1, the nuclear volume of group X3Y0 is smaller than that of group X6Y0 and group X9Y0. In EG2, the nuclear volume of group X6Y- is smaller than that of group X6Y6 and group X6Y0. It is speculated that the volume of the nucleus is affected by the vertical compression of actin stress fibers and the lateral tensile force. The cells in the X3Y0 and X6Y- groups have less of both forces, so the cell nuclear volume is smaller.

Effect of nuclear thickness, volume, and surface on YAP’s expression

YAP is a key molecule in the Hippo signaling pathway. It is involved in regulating the biological functions of cell proliferation, survival, differentiation, and organ development. YAP constantly shuttles between the nucleus and cytoplasm, and its activation requires their accumulation in the nucleus [39,40]. Researchers have found that pressure on the nucleus can induce the transfer of YAP from the outside to the inside of the nucleus [41]. The analysis of three-dimensional nuclear images showed that, in both EG1 and EG2, the total amount and the average fluorescence intensity of YAP expression in the nucleus were the highest in the X6Y0 group, while the average fluorescence intensity of YAP in the cytoplasm was relatively low, indicating the accumulation of YAP in the nucleus of X6Y0 group, which promoted cell proliferation. In the experiment, the X6Y0 group was also observed to have the smallest nuclear thickness, demonstrating that the nucleus of this group was subjected to greater pressure. Nuclear pressure can enlarge the nuclear pore and reduce the mechanical constraint on YAP nuclear displacement [41,42]. According to the data trends of nuclear volume and surface area, it is speculated that the surface area of the nucleus has a greater influence on YAP’s entry into the nucleus than the volume. The expansion of the nuclear surface area may lead to the opening of the nuclear pore and promote the entry of YAP.

Effect of different loading types on cell arrangement

It was found that cells tended to line up perpendicular to the direction of strain ^[5,42]. Previous studies have shown that Cells tend to orient in the direction where the maximum density of stress fibers, and hence the strongest cell-substrate attachment, can be achieved. They found that for a cell-substrate system subjected to cyclic stretch, the final alignment of cells represents the competitive coupling between stress fibers [43]. The rearrangement of cells under the influence of mechanical factors is of great significance for the correct formation of tissues and the healing of wounds [5,44,45]. In this study, it was found that the arrangement of cells was not affected by mechanical strain when the cells were subjected to isotropic stretch in group X6Y6. When subjected to anisotropic mechanical stimulation, the cells tend to line up perpendicular to the direction of the greatest strain. In the X6Y0 group, the strain in the Y direction was basically zero, and the cell arrangement was faster and better. Therefore, the direction and time of cell arrangement can be controlled by changing the strain stimulus in different directions.

Conclusion

Mechanical stimulation affects the proliferation, differentiation, and expression of osteoblasts. Currently, many studies about the effect of stretch on osteoblasts focused on the frequency, time, and size of tensile stress, ignoring the effect of loading types.

In this study, cyclic loading with varied strain and negative ratio of transverse strain to axial strain were applied to osteoblasts at a frequency of 1 Hz and a periodicity of 1 h/day. It was found that the loading condition of 6% strain and 0 NR would most promote cell proliferation. This condition thickens the F-actin cytoskeleton, increases the pressure on the nucleus, and further increases the accumulation of YAP in the nucleus. Cyclic strain stimulation with different NR also affected cell arrangement. Through this study, we can further understand the mechanism of cell mechanical sensitivity, and guide the application of structures with different Poisson’s ratio in bone tissue engineering.

Acknowledgments

This work was supported by the [National Key Research and Development Program of China #1] under Grant [number 2023YFC2410404]; [National Natural Science Foundation of China #2] under Grant [number12172034, U20A20390, 11827803]; [Beijing Municipal Natural Science Foundation #3] under Grant [number 7212205]; [111 project #4] under Grant [number B13003]; and [Fundamental Research Funds for the Central Universities #5].

Disclosure statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19475411.2024.2338737.

Additional information

Funding

The work was supported by the Beijing Municipal Natural Science Foundation [7212205]; National Key Research and Development Program of China [2023YFC2410404]; National Natural Science Foundation of China [12172034, U20A20390, 11827803]; Fundamental Research Funds for the Central Universities; 111 project [B13003].