Response of bone marrow derived connective tissue progenitor cell morphology and proliferation on geometrically modulated microtextured substrates

Abstract

Varying geometry and layout of microposts on a cell culture substrate provides an effective technique for applying mechanical stimuli to living cells. In the current study, the optimal geometry and arrangement of microposts on the polydimethylsiloxane (PDMS) surfaces to enhance cell growth behavior were investigated. Human bone marrow derived connective tissue progenitor cells were cultured on PDMS substrates comprising unpatterned smooth surfaces and cylindrical post microtextures that were 10 μm in diameter, 4 heights (5, 10, 20 and 40 μm) and 3 pitches (10, 20, and 40 μm). With the same 10 μm diameter, post heights ranging from 5 to 40 μm resulted in a more than 535 fold range of rigidity from 0.011 nNμm⁻¹ (40 μm height) up to 5.888 nNμm⁻¹(5 μm height). Even though shorter microposts result in higher effective stiffness, decreasing post heights below the optimal value, 5 μm height micropost in this study decreased cell growth behavior. The maximum number of cells was observed on the post microtextures with 20 μm height and 10 μm inter-space, which exhibited a 675 % increase relative to the smooth surfaces. The cells on all heights of post microtextures with 10 μm and 20 μm inter-spaces exhibited highly contoured morphology. Elucidating the cellular response to various external geometry cues enables us to better predict and control cellular behavior. In addition, knowledge of cell response to surface stimuli could lead to the incorporation of specific size post microtextures into surfaces of implants to achieve surface-textured scaffold materials for tissue engineering applications.

Fig. 1

PDMS post microtextures with varying geometry and arrangement. Cylindrical post microtextures that were 10 µm diameter, 4 different heights (5, 10, 20 and 40 µm) and 3 different inter-spaces (10, 20, and 40 µm) were (a) designed. (b) Theoretical total surface area of post microtextures in 100 µm × 100 µm square.

Fig. 2

Control the thickness of SU-8 layers. (a) To obtain appropriate micropost heights (5, 10, 20 and 40 µm), SU-8 2010 was spin coated onto a silicon wafer with an optimal speed. (b) Cross-section of SU-8 which was spin coated onto silicon wafer. Thickness of the SU-8 layers was verified using SEM.

Fig. 3

Pattern layout on the photomask for the pattern with varying inter-space (10, 20 and 40 µm) dimensions between posts. Dark regions correspond to chrome (opaque) regions on the photomask, and light regions correspond to regions transparent to UV exposure.

Fig. 4

Fabrication of PDMS post microtextures by soft lithography. Briefly, various film thicknesses (5, 10, 20 and 40 µm) of SU-8 2010 photoresist were spin coated on top of silicon wafers at different optimal speeds. By using UV exposure, the post microtexture patterns with varying inter-space (10, 20 and 40 µm) dimensions were transferred from a photomask onto the photoresist, and then developed. The liquid PDMS were mixed at a ratio of 10:1, degassed for 20 min, and then poured uniformly on top of the patterned mold. The PDMS substrates were cured at 85°C for 2 h.

Fig. 5

SEM images of PDMS post microtextures of 10 µm diameter, and 5 µm, 10 µm, 20 µm, and 40 µm heights with the 10 µm, 20 µm, and 40 µm separation (IS=inter-spaces) between the posts.

Fig. 6

PDMS micropost arrays to engineer substrate stiffness. (a) Graphical depiction of finite-element method (FEM) analysis of micropost of heights (H) each bending in response to applied horizontal traction force (F) of 20nN. (b) Normal spring constant (K) as a function of H, as computed from FEM analysis (bars) and from Euler-Bernoulli beam theory (curve). K measures micropost stiffness (rigidity).

Fig. 7

SEM images showing morphology of cells grown on the PDMS post microtextures that are 10 µm diameter, (a) 5 µm, (b) 10 µm, (c) 20 µm, and (d) 40 µm heights with the 10 µm, 20 µm, and 40 µm separation (IS=inter-spaces) between the posts. On Day 9, cells on post microtextures tended to attach next to the posts and spread between them while directing their processes towards posts and other cells (white arrows). Regardless post heights, the narrowest processes were observed on cells cultured on the post microtextures with 10 µm IS, in which individual cells tended to grow between and along the array of posts. On the smooth surfaces and post microtextures with 40 µm IS, cell bodies adopted a broad flattened shape and appeared to anchor to random locations on the surface as they migrated.

Fig. 8

Cell morphology with PDMS post microtextures that are 10 µm diameter (D), 10 µm and 40 µm inter-space (IS) between the posts with the 10 µm and 40 µm heights (H). (a) CTPs on 10 µm height of post microtextures with 10 µm inter-spaces interact with the bottom of the microtextures as well as with the sides. Cells tended to attach next to the posts and spread between and top of them, exhibited highly contoured morphology, and directed their long processes towards posts and other cells. (b) On the 10 µm height micropost with 40 µm inter-spaces, cells are likely to have contact to only one micropost side and the bottom at the same time. The morphology of cells grown on the microposts showed to anchor to random locations and cell bodies adopted a broad flattened shape. (c) Cells on post microtextures with 40 µm heights and 10 µm inter-spaces attach and spread on the top of the them, and pulled posts around cells. (d) Cell morphology on post microtextures with 40 µm heights and 40 µm inter-spaces bent posts around cells and exhibited similar shape to cells on smooth surfaces that appeared to anchor to random locations on the surface as they migrated and cell bodies adopted a broad flattened shape.

Fig. 9

Fluorescent microscopy (Red) and bright-field (Grey) images of CTPs near the interface between a smooth surface and PDMS post microtextures that are 10 µm diameter, the 10 µm, 20 µm, and 40 µm separation (inter-spaces: IS) between the posts with the (a) 5 µm, (b) 10 µm, (c) 20 µm and (d) 40 µm height. More cells attached and proliferated on the post microtextures compared to cells on smooth surfaces. On Day 9, more cells stained intensely for DAPI (yellow) and AP (red) on the post microtextures compared to cells on smooth surfaces.

Fig. 10

CTP proliferation on PDMS post microtextures and corresponding smooth surfaces after 9 days of culture. (a) All post microtextures had increased numbers of cells compared to the smooth surfaces. The maximum number of cells was observed on the post microtextures with 20 µm height (H) and 10 µm inter-space (IS) (20H-10IS), compared with the number of those on the smooth surfaces (p < 0.05). For the same height, post microtextures with 10 µm inter-space have shown highest cell numbers. For the same inter-space, post microtextures with 20 µm height have shown highest cell numbers. (b) Post microtextures with 10 µm height and 10 µm inter-space (10H-10IS) have shown cell numbers normalized to surface area that are nearly three times greater than that on the smooth surface, while 40 µm height and 20 µm inter-space (40H- 20IS) post microtextures exhibited the lowest cell number/unit area. For the same height, post microtextures with 10 µm and 40 µm inter-spaces have shown higher cell numbers while 20 µm inter-space have shown lowest cell numbers. For the same inter-space, post microtextures with 40 µm height have shown lowest cell numbers. * denotes statistical significance compared to other surfaces (p < 0.05).