Engineered barriers regulate osteoblast cell migration in vertical direction

Engineered barriers with guiding patterns

In order to study the vertical migration of MC3T3 cells, PDMS platforms with increasing heights of 1, 5, 10, and 25 µm barriers were fabricated. Scanning electron micrographs of 5 and 25 µm tall barriers with a grating of 5 µm width, 5 µm spacing, and 1 µm thick are shown in Fig. 2a,b. As cells on flat surfaces migrate randomly and cells on grating migrate along the grating either towards or away from the barriers, it is necessary to form patterns to guide cells towards the barriers. Thus, arrows with confined channels were fabricated for guidance. The arrows pattern with a 45° angle, 10 µm width, and 26 µm length with 26 µm spacing between arrowheads in 12 µm tall, 20 µm wide channel walls, 10 µm wide channels between channel walls, and 25 µm tall barrier is shown in Fig. 2c. Moreover, to promote cell migration up the barriers, PDMS barriers with slope angles of 18° and 40° were fabricated. Figure 2d shows the 18° sloped sidewall and 10 µm tall barriers that extend 35 µm from the top of the barrier to the bottom next to a grating pattern.

Effects of barrier height and guiding pattern on vertical cell migration

When MC3T3 cells migrate and encounter a barrier, they will move in three different ways: climb vertically to the top of the barrier, move along the sidewalls of the barrier, or turn back. These three movements are shown in Supplementary Video SV1. The probability of MC3T3 cells climbing up vertically to the top of the barriers is shown in Fig. 3a. When cells migrated from a flat surface or grating towards the barriers, the probability of cells climbing up decreased with increasing barrier heights. When the barrier height was increased from 1 to 25 µm, the probability of cells climbing up the barrier decreased from 39.3% to 2.7% and from 56.0% to 10.7% for flat surface and grating, respectively. The higher probability for cells to climb up when they started on a grating than a flat surface suggests that the grating guiding pattern promoted vertical migration of cells. It is well-known that gratings can accelerate cell migration speed and enhance migration directionality^37,40, making it easier for cells to migrate vertically.

When the barrier height was 1 µm, the vertical climbing probability for cells guided by arrows in channels was lower than cells that started on a flat surface or grating. This low climbing probability may be due to cell interactions in the 10 µm wide channels. When MC3T3 cells migrated in the 10 µm wide channels, they elongated and adhered on both the bottom and sidewalls of the channels. Some of the cells migrated along the channel sidewalls and climbed up to the top of the channel walls. These cells needed to climb down the 11 µm tall channel walls before reaching the 1 µm tall barrier. As a result, the probability of cells climbing over the 1 µm tall barrier for cells guided by arrows in channels was lower than those guided by a flat surface or grating.

The overall vertical climbing probability increased to 33.5% when the barrier height was 5 µm, most similar to the probability of cells climbing up when guided by a grating. The vertical climbing probability decreased when the barrier height was further increased, with cells from the flat surface or grating showing the same trends. The vertical climbing probability of cells on arrows in channels was higher than cells on flat surface or grating when barriers were 10 µm or taller. The vertical climbing probability for cells in channels with arrows was 30.8% and 15.2%, more than 40% higher than cells that started on grating when the barriers were 10 and 25 µm tall, respectively. The enhancement of vertical climbing for cells guided by arrows in channels can be attributed to the improvement of migration directionality due to confinement by the channel sidewalls in the 10 µm wide channels. As cells could only climb up or reverse migration direction when guided by arrows in channels, the vertical climbing probability increased by such guiding pattern.

MC3T3 cells climbing down barriers were also studied in addition to cells climbing up. As shown in Fig. 3b, the ratio of cells climbing up/climbing down is usually larger than 1, meaning that it was easier for cells to climb up than to climb down the barriers. When the barrier was 1 µm tall, the ratio of cells climbing up/climbing down was about 1, indicating that nearly the same number of cells climbed up or down the 1 µm tall barrier. When the barrier height was increased to 5 µm, it was more difficult for cells to climb down to the flat surface compared to when the bottom surface had a grating or arrows in channels pattern. The presence of a 1 µm thick grating or arrows in channels reduced the height difference from the top of the barrier to the bottom surface. It provided more adhesion areas, thus reducing the difficulty for cells to climb down from the top of the 5 µm barrier to the bottom. For 10 µm tall barriers, fewer cells could reach the bottom surface from the top of the barrier. As a result, guiding patterns with grating or arrows in channels showed fewer cells climbing down the barriers than climbing up. When the barrier height was further increased to 25 µm, both climbing up and climbing down became more difficult for the cells. The ratio was reduced to 2.2 and 1.6 for grating and arrows in channels on the bottom surface, respectively. When the bottom was a flat surface, no cells could climb down from the 25 µm tall barrier. On the contrary, a few cells could still climb up from the bottom to the top of the 25 µm tall barrier.

In general, it is more difficult for MC3T3 cells to climb down than climb up the barriers. This may be due to the difficulty for cells to move over the convex angle between the top surface and the barrier sidewall compared to the concave angle between the bottom surface and the sidewall. Several papers have reported how surface curvature regulates cell migration^{41,42,43,44,45}. It has been found that cells on a concave surface migrate faster than when they move on a convex surface. Adherent cells prefer to position themselves on a concave surface and avoid migration through convex areas. On convex surfaces, cells fully make contact around the curvature, causing substantial nuclear deformation. On the contrary, cells do not make full contact around the concave curvature. Therefore, MC3T3 cells could climb up the barrier more easily by reaching the barrier sidewall around the concave curvature. Dapi/Hoechst staining of cells on platforms can be used to quantify nuclear deformation on convex or concave surfaces. However, stained cells are static, and they lack the dynamic information needed to distinguish whether the cells are migrating up or down the barriers. Instead, a high-resolution confocal microscope will be used to dynamically track the cell migration around the barriers in the 3D imaging mode in future study to provide more insights in the changes of cell morphology.

MC3T3 cell migration speed and directionality on engineered patterns

In order to better understand the cell guiding effects of grating and arrows in channels, cell trajectory, speed, and directionality on these patterns were studied. As shown in Fig. 4a, trajectories of MC3T3 cells on flat surfaces show no directional preference, resulting in random movements. On surfaces with a grating, cells migrated along the y-axis, which corresponds to the grating orientation; this is caused by the guiding effect of gratings on cell migration. Trajectories of cells on the surface with arrows show weak guidance along the arrow direction, while arrows in channels effectively guided the cells along the channels. Cells migrated furthest on the surface with arrows in channels, covering a distance of about 510 µm, while they covered about 370 µm on the flat surface and surface with grating or arrows.

The migration speed of MC3T3 cells on different patterns is shown in Fig. 4b. It was found that cells on arrows in channels migrated fastest, with an average speed of 0.53 μm/min. This is 36% faster than cells on a flat surface, 18% faster than on arrows, and 8% faster than on grating. From the directionality results shown in Fig. 4c, alignment angles for flat surface, grating, arrows, and arrows in channels are 44.6°, 56.1°, 56.5°, and 70.1°, respectively. A larger angle indicates that cells were better guided along the pattern orientation. These results show that cells that migrated on arrows in channels exhibited the best directionality, cells on grating and arrows showed a similar guiding effect, and cells on flat surface migrated randomly. The cell migration direction on a flat surface and grating match previous studies^37,40,46. The faster and more directional migration of MC3T3 cells on surfaces with grating and arrows in channels helps them climb up taller barriers.

In our previous published work³⁷, we focused on the effects of patterned features on osteoblast cell migration on 2D platforms. The controlled patterns included gratings with various depths, bending angles, densities of grating with bends, and discontinuous gratings. In this work, patterned gratings and arrows were used as guiding patterns to promote cell migration over barriers with different heights that were perpendicular to the cell migration direction. These barriers also had various slope angles, and cell migration up or down these barriers was studied. Our previous work focused on enhancing cell migration on 2D patterns, while the main objectives of this work are to investigate 3D cell migration across vertical and sloped barriers.

Focal adhesions (FAs) connect the actin cytoskeleton to the substrate or extracellular matrix, to transmit forces to the substrate. Dynamic FA affects cell adhesion and motility. According to previous works¹⁷, it was found that with guidance of patterns such as gratings, cells prefer aligning along with gratings, and FA mostly aligns to gratings as well. Cells tend to migrate faster with better directionality with guiding of topographic cues compared with on flat surfaces³⁴. When cells migrate towards the barrier with guidance of topographic cues like gratings, cells tend to elongate along the gratings with asymmetric FA on leading edge and trailing tail, which explores and senses the extracellular environment, takes place primarily around the cell front⁴⁷. When cells encounter the vertical barrier, more projection of forward lamellipodia and filopodia can better sense topographic cues from the barrier rather than hindered by the barrier sidewalls immediately. In other words, the more lamellipodia and filopodia sense the barrier, the easier for cells to climb over the barrier. Therefore, directional migration may drive more climb up to the vertical barriers.

MC3T3 cell migration in vertical direction

Micrographs of MC3T3 cells on grating next to barriers with different heights were investigated to study how cells migrate vertically. Figure 5a shows a MC3T3 cell elongated along the grating, climbing vertically over the 1 µm tall barrier. When the barrier height was increased to 5 µm, the trailing part of the MC3T3 cell still aligned to the grating while the leading part climbed onto the top of the barrier with a smaller angle long the sidewall compared to the 1 µm tall barrier, as shown in Fig. 5b. When the barrier height was further increased to 10 µm, the MC3T3 cell reached the top of the barrier at a slight angle along the sidewall, as shown in Fig. 5c. With a 25 µm tall barrier, as shown in Fig. 5d, the MC3T3 cell moved along the sidewall of the barrier obliquely to reach the top of the barrier. Cell migration is driven by lamellipodia extensions, which push the cell forwards⁴⁸. When a cell encounters a barrier, the extension of lamellipodia is hampered. Some cells may only contact part of the barrier sidewall when barriers are taller, as shown in Supplementary Fig. S1. As cells need to make surface contact to migrate, insufficient surface contact may make it difficult for cells to reach the top of the barriers⁴⁹. In order to climb up the taller barriers, cells must make full contact with the sidewall, which may cause them to prefer to climb obliquely along the barrier sidewall rather than vertically up the taller barriers.

The time-lapse imaging videos as shown in supplementary videos SV2 and SV3 provided dynamic information for how cells climbed up or down the barriers. The climbing angle along the sidewalls of the barriers was calculated, as shown in Fig. 6a. It was found that when the barrier was only 1 μm tall, cells climbed up perpendicular to the barriers. When the barrier height was increased to 5 μm, the climbing angle decreased for cells on a flat surface or grating. When the barrier height was further increased to 25 µm, the climbing angle decreased to 10.3° for cells started on a grating, meaning that the cells climbed along the barrier sidewall. For cells on the surface with arrows in channels, the sideways movement of cells was limited by the channel walls, and the climbing angle was large when the barrier height was 10 μm or less. When the barrier was 25 μm tall, exceeding the 12 μm tall channel walls, some cells moved to the top of the channel walls. Overall, cells from the surface with arrows in channels could climb to the top of the 25 μm tall barrier with a smaller climbing angle of 32.3°, compared to cells climbing over a 10 μm tall barrier with a larger climbing angle of 82.9°.

In addition to the climbing angle, climbing time also indicates the degree of difficulty for cells to climb over a barrier. Climbing time is defined as the time from when a cell touches the bottom of the barrier until the entire cell body reaches the top of the barrier. Figure 6b shows that climbing time increased with taller barriers for cells that started on a flat surface or grating. The results are consistent with the micrographs shown in Fig. 5 and climbing angles shown in Fig. 6a. When the barrier height was low, it took only a short amount of time for cells to climb up to the top of the barriers. For taller barriers, cells contacted the barrier sidewall first and gradually climbed up until its entire body reached the top of the barriers, which resulted in a longer climbing time. For cells that started from a surface with arrows in channels, climbing time varied with barrier height. This may be related to the varying distance between the top of the channel wall and the top of the barrier. Some cells that started from the surface with arrows in channels could travel to the top of the 12 μm tall channel walls. For cells on top of the channel walls, to get over the 1 μm tall barrier, cells needed to climb down 11 μm to get over the 1 μm tall barriers. Likewise, to get over the 10 μm tall barrier, cells needed to climb down 2 μm to the top of the 10 μm tall barriers. Therefore, the probability of getting over the barriers varied due to some cells that climbed on top of the channel walls, which made the distance between the top of the channel walls and the barriers vary with 11, 7, 2, and 13 μm when the barrier height varied from 1, 5, 10, and 25 μm, respectively.

MC3T3 cell migration modes up and down vertical barrier

Fluorescence micrographs of MC3T3 cells migrating up and down a 10 µm tall barrier were shown in Figs. 7 and 8, respectively. Projected fluorescence signal, together with the 3D reconstruction of the cell morphology and sideviews of the cells located across the barriers were displayed.

The process of cells moving up or down the 10 µm tall barrier can be shown in 4 steps. When the cell reached the barrier from the bottom surface, the leading edge was aligned to the edge of the barrier as shown in Fig. 7a. The cell then adhered to the top surface. Meanwhile, the adhesions on the sidewall and on part of the bottom surface were released, changing the cell into an elevated morphology as shown in Fig. 7b. The cell did not contact the sidewall conformally, leaving a gap between the sidewall and the cell membrane. The cell then aligned itself and proceed to migrate up the barrier while keeping the elevated morphology as shown in Fig. 7c. In the end, cell nucleus moved up the barrier. Due to the elevated cell morphology, the nucleus was only slightly deformed when crossing the barrier as shown in Fig. 7d.

Cells moving down the barrier followed a similar migration pattern, but the cell morphology during the process was different. Leading edge of the cell aligned to the barrier edge and reached down to the bottom surface as shown in Fig. 8a,b, but the cell membrane conformed to the vertical sidewall. When the nucleus moved down the barrier, it was significantly deformed to fit the shape of the barrier sidewall as shown in Fig. 8c. When the cell body migrated down the barrier to the bottom surface, the trailing edge was still conformed to the sidewall as shown in Fig. 8d.

It was also noted that cells moving up the barrier have clearly defined actin stress fibers during the process. But when cells moved down the barrier, the stress fibers in the leading edge were disrupted. It is suggested that cell migration directionality will be affected due to the deformed cell morphology and disrupted actin stress fibers, making the cells harder to move down a barrier.

Sloped barrier promoted vertical migration

The effects of a sloped barrier on vertical cell migration were studied. As mentioned in Fig. 3a, when the barrier was 10 µm tall, only 20.6% of MC3T3 cells could climb up to the top of the barrier with the help of a grating pattern. Based on this result, sloped barriers with 18° and 40° sidewalls were fabricated next to the 10 µm tall barrier with grating guidance. It was found that 10 µm tall barriers with an 18° sloped sidewall increased the probability of MC3T3 cells climbing up to the top of the barrier from 20.6% for a vertical barrier to 40.6% for a barrier with 18° sloped sidewall, a 97.0% increase as shown in Fig. 9a. Likewise, the probability of MC3T3 cells climbing down from the top of the barrier to the bottom surface increased from 3.4% for the vertical barrier to 20.3% for the 18° sloped barrier, five times the original value. Therefore, barriers with sloped sidewalls can significantly promote the vertical migration of MC3T3 cells.

Micrographs of MC3T3 cells climbing 10 µm tall, 18° and 40° sloped barriers are shown in Fig. 9b,c, respectively. The MC3T3 cell body crossed the 10 µm tall, 18° sloped barrier with the shortest distance. When the barrier had a 40° sloped sidewall, the cell body crossed the barrier sidewall at a small angle. As shown in Fig. 6a, the smaller the climbing angle, the lower the probability for MC3T3 cells to climb up or down when they migrated vertically. As a result, barriers with sloped sidewalls assist the vertical migration of MC3T3 cells.