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. Author manuscript; available in PMC: 2012 Jan 1.
Published in final edited form as: Soft Matter. 2011 Jan 1;7:3599–3606. doi: 10.1039/C0SM01353F

Human macrophage adhesion on polysaccharide patterned surfaces

Irene Y Tsai b, Chin-Chen Kuo b, Nancy Tomczyk a, Stanley J Stachelek c, Russell J Composto b, David M Eckmann a,*
PMCID: PMC3072250  NIHMSID: NIHMS269664  PMID: 21479122

Abstract

Despite many advances in designing biocompatible materials, inflammation remains a problem in medical devices and implants. We report two methods, microcontact printing and photodegradation by UV exposure, to pattern dextran and hyaluronic acid on glass, as well as demonstrate their utility for use as an anti-inflammatory biomaterial. The dextran/glass patterned surface can be further modified by grafting hyaluronic acid to glass, creating a binary polysaccharide patterned surface. We used two geometries, 90 µm squares and 22 µm stripes, to study the human macrophage (THP-1) adhesion on the patterned surfaces containing dextran, hyaluronic acid and the binary pattern. The results indicate that a majority of the macrophages are non-adherent on hyaluronic acid for three day culture. The ranking of surfaces according to macrophage adhesion is 3-aminopropyl triethoxysilane-modified glass culture dish, dextranized surfaces, glass, and hyaluronic acid-modified surfaces. On the binary pattern of dextran and hyaluronic acid, macrophages preferentially attach and adhere to the dextranized area. Patterned surfaces provide an excellent platform for mimicking the complexity of the glycocalyx and investigating the interface between this surface and cells. This binary polysaccharide pattern also offers a new route to address anti-inflammatory potential of surface coatings on biomaterials in a high through-put fashion.

Introduction

The endothelial glycocalyx layer is the surface layer of the vascular endothelium. This layer consists mostly of carbohydrates and has several important functions such as regulating the vascular permeability1,2, acting as a mechanotransducer3 and balanced signaling events in the local microenvironment4. During inflammation, the glycocalyx layer has been shown to degrade to facilitate leukocyte recruitment5,6. Conversely, an undamaged layer inhibits a cascade of inflammatory events, and thus, mimicking the endothelial glycocalyx surface can be a route to reduce inflammation caused by medical implants and devices7. Here, we report the adhesion of the human macrophage cell line, THP-1, on surfaces presenting immobilized dextran, hyaluronic acid, patterns of dextran and hyaluronic acid on glass, as well as binary patterns of dextran and hyaluronic acid. These surfaces profile a model platform for better understanding the anti-inflammatory potential of the glycocalyx.

Because the endothelial glycocalyx layer is heterogeneous, composed of different polysaccharides5, and rough, it is conceivable that the spatial organization of the polysaccharides and the topography play an important role in the adhesion of macrophages, and consequently, their inflammatory responses. Wójciak-Stothard etal.8 studied P388D1 macrophages on microfabricated grooves (30 to 282 nm) and found that cytoskeletal organization and focal adhesion depended on the nano-sized features. They also found more phagocytotic activity on the patterned surfaces than fused silica substrata8. In addition, Zhang’s group found mouse macrophages adhere only to the gold regions of a surface patterned with PEG9.

Anderson’s group has several studies using human peripheral blood monocytes on surfaces of different hydrophilicity and charges10, as well as photochemically micropatterned surfaces11. This group found that significantly more adherent macrophages experience apoptosis on the hydrophilic and anionic surfaces than the hydrophobic or the cationic surfaces in vitro12 and in vivo13. In terms of cytokine responses, Brodbeck et al.12 showed human macrophages express more interleukin-10 (anti-inflammatory) and less interleukin-8 (pro-inflammatory) on hydrophilic and anionic surfaces, respectively, indicating that surface modification is directly related to inflammatory response. While these experiments confirm the importance of surface chemical heterogeneity and surface topography on macrophage adhesion and activity, few experiments examine how surface coating containing the molecular constituents of the endothelial glycocalyx can reduce inflammatory responses provoked by biomaterials. In this study, we vary the spatial distribution of the dextran and hyaluronic acid on a surface by using microcontact printing and photochemical micropatterning to investigate human macrophage adhesion. Insights from these studies can lead to improved coatings that reduce inflammation on medical implants, biosensors and other medical devices.

Surface patterning has become an important route to fabrication of many biomedical technologies such as biosensors14, smart biomaterials15, tissue engineering16 and microfluidic devices17. Several techniques are available to pattern a surface having nano- to micron-size topographies, differing degrees of hydrophobicity and various biomolecular ligands. In particular, microcontact printing and photochemical micropatterning are facile techniques that do not require elaborate cleanroom facilities. Microcontact printing has been used widely to pattern proteins18,19, polysaccharides20,21, DNA22 and cells23,24. For example, Zhou et al. used microcontact printing to pattern tetraoctadecylammonium bromide on carboxymethylated dextran hydrogels for protein microarray applications25. Furthermore, surface patterning of hyaluronic acid on a poly(ethylene terephthalate) film has been shown to be effective in antithrombogenicity26. Others have used photochemical micropatterning to pattern proteins by photodegradation of a poly(oligo(ethylene glycol) methacrylate) brush27. Lu et al.28 studied cell adhesion of fibroblasts and immortalized cancer cells on patterned polyelectrolyte multilayers manufactured using a room-temperature imprinting method and demonstrated that local geometry of patterned stripes (e.g., height and width) regulates cell attachment.

Although microcontact printing appears to have become a routine method18–25 the combination of pattering HA and dextran has not previously been achieved and is demonstrated successfully herein. This has particular significance for vascular biology applications specifically because these two different polysaccharides are primary components of the endothelial cell glycocalyx layer which is in direct contact with circulating blood. We used microstamping and photodegradation methods to fabricate five different patterned surfaces, namely, (1) dextran and glass (dextran/glass), (2) hyaluronic acid and glass (hyaluronic acid/glass), (3) dextran and 3-aminopropyl triethoxysilane-modified glass (dextran/APTES-glass), (4) hyaluronic acid and APTES modified glass (hyaluronic acid/APTES-glass), as well as (5) dextran and hyaluronic acid (dextran/hyaluronic acid). We also used exemplars of patterned surfaces to study related cell adhesion interactions with macrophages.

To study macrophage adhesion, we chose patterned surfaces generated by the photodegradation method. Patterned surfaces of rhodamine-dextran/glass, fluorescein-hyaluronic acid/glass and rhodamine-dextran/hyaluronic acid were used to investigate how micrometersized patterns direct macrophage adhesion. The patterned surfaces were characterized by fluorescence microscopy and atomic force microscopy, and their corresponding surface chemistries were also verified with contact angle and ellipsometry measurements of the homogeneous surfaces. To understand the macrophage adhesion on patterned surfaces, we quantified the macrophage spreading area on homogeneous surface of dextran (or rhodamine-dextran), hyaluronic acid (or fluorescein-hyaluronic acid), glass, APTES-glass and culture dish. We found that macrophages have the highest spreading area on APTES among the surfaces we studied, followed by culture dish, dextran, glass and hyaluronic acid surfaces. This result is consistent with the patterned surfaces. In particular, the binary polysaccharide surface consisting of dextran and hyaluronic acid show that macrophages preferentially adhere to the dextranized regions. This patterned surface can be used as a model to understand the interaction between the macrophages and the endothelial glycocalyx surface. Furthermore, this preferential nonattachment and non-spreading onto the hyaluronic acid can be exploited to surface-direct cellular responses in applications for medical devices and therapeutic and diagnostic tools.

Materials and methods

All solvents and reagents were utilized as received. 3-Aminopropyl triethoxysilane (APTES), sodium periodate (NaIO4), sodium cyanoborohydrate (NaBH3CN), 1, 6-diaminohexane, dimethylformamide and HEPES were purchased from Sigma-Aldrich Co. and Fisher Scientific. Dextran is from Leuconostoc mesenteroides with molecular weight of 64,000–76,000 (Sigma # D4751). Hyaluronic acid has molecular weight of 750,000, Sigma #H1504). For fluorescence imaging, rhodamine B isothiocyanate-dextran (Sigma #R9379, MW=70,000) and fluorescein hyaluronic acid (Sigma #F1177, MW= 800,000) are used. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and sulfo-NHS were purchased from Thermo Fisher Scientific, #22980 and #24510, respectively. Sylgard 184 Silicone elastomer curing agent and elastomer base were purchased from Dow Corning to make poly(dimethylsiloxane) (PDMS) stamps. Silicon oxide surfaces were purchased from Silicon Quest International (Santa Clara, CA). Two different TEM grids were used, Electron Microscopy Science, Cat# G200-Cu and Cat # G400P-Ni.

Microcontact printing on glass or PU films

The PDMS stamp was prepared by casting the mixture of elastomer curing agent and elastomer base (ratio 1:10) against a patterned silicon wafer (master). The master patterned surface (silicon) consists of 10 µm stripes. Oxidized rhodamine dextrans were prepared using the same procedure described in previous publication29. A drop of oxidized dextran (100 µl) was pipetted onto the patterned surface of the PDMS and covered for 1 hour. For fluorescence imaging, rhodamine dextran was used in place of dextran. Dextran was then removed from the patterned surface and let dry for 1 minute before stamping the patterns onto an aminated glass or an aminated PU film. The glass or a PU film was aminated via APTES or 1, 6-diaminohexane, respectively. Gentle pressure was applied to the PDMS to make sure that the patterns were in contact with the substrate. The substrate was covered with aluminum foil overnight before the stamp was peeled away, yielding a surface having a pattern of rhodamine dextran/APTES-glass or dextran/NH2-PU. The surface was rinsed several times with de-ionized water.

Photodegradation micropatterning on glass

Silicon wafers and glass coverslips were cleaned using “piranha” solution (70% H2SO4 and 30% H2O2, v/v) for 20 minutes at 80 °C to ascertain a homogeneous silanol layer that could react with APTES. The surfaces were washed with water numerous times and dried in an oven. Upon a dried clean silicon surface, the silane APTES reaction was carried out immediately. The APTES thickness and contact angle were verified by ellipsometer (Rudolph AutoEL II) and contact angle goniometer, respectively. A TEM grid (EMS # G400P-Cu & G200-Cu) was used as a photomask. The APTES surface was exposed to UV for 7 minutes. The unmasked APTES regions were degraded by UV. This produced a silanol and amine functionalized patterned surface. The surfaces were washed with toluene and water several times.

The silanol/aminated glass substrates were immersed in aqueous oxidized dextran solution. The oxidation conditions and the extent of oxidation were studied previously (#). Briefly, dextran solution (2 mg/mL) was prepared and oxidized using 1.5 gram of NaIO4 in the reaction. After oxidizing for 1 hour, NaBH3CN (15 mg) was dissolved into the oxidized solution. The reaction was placed on an orbital shaker for 1 day. The dextran patterned surface was removed from the solution and washed with water several times. For hyaluronic acid patterning, a well established procedure, the EDC-mediated condensation with N-hydroxysuccinimide, was followed. Specially, the silanol/aminated glass substrate was immersed in a solution containing hyaluronic acid (2 mg/ml), EDC (38.2 mg/ml), Sulfo-NHS (10.8 mg/ml) and HEPES (2.3 mg/ml) for 1 day at room temperature. After one day reaction, the hyaluronic acid patterned surface was washed with water many times and dried using nitrogen gas.

Binary polysaccharide patterns

Subsequent to successfully patterning of dextran/glass or dextran/APTES-glass, hyaluronic acid (or fluorescein-hyaluronic acid) was immobilized onto the non-dextran regions (Figure 1). Rectangular pattern of dextran on glass via photodegradation micropatterning or microcontact printing was immersed in hyaluronic acid (2 mg/ml), EDC, HEPES and Sulfo-NHS, following a procedure described earlier. After two day reaction, the dextran/hyaluronic acid patterned surface was washed with water many times and dried using nitrogen gas.

Figure 1.

Figure 1

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Schematic representation of rhodamine-dextran patterns fabricated by (A) microcontact printing and (B) photodegradation of APTES and subsequently reacted with fluorescein-hyaluronic acid.

Surface characterization

Homogenous surfaces of dextran or hyaluronic acid were verified using contact angle and ellipsometry when appropriate. The fluorescence images were taken using an Olympus BX50 microscope to verify the success of the patterned surfaces. The patterned surfaces were characterized using scanning force microscopy (Veeco) in both dry (air) and wet (PBS buffer solution) conditions. The images were analyzed to obtain the degree of swelling for dextran and hyaluronic acid.

Macrophage culture

The human monocyte derived macrophage cell line, THP-1 (ATCC Manassas, VA), were grown in RPMI medium (Cell Culture Technologies, Herndon VA) supplemented with 5% fetal calf serum and 0.05 µM 2-mercaptoethanol. To assess inflammatory cell attachment to the patterned surfaces, the THP-1 cells were stimulated with the addition of 1.6 × 10−7 M phorbol 12-myristate 13-acetate30 and seeded onto the modified surfaces at a concentration of 500,000 cells/ml. Macrophages were cultured on the substrates for 1, 2 and 3 days. Macrophages on homogeneous surface were imaged using optical microscopy. For the patterned surfaces, an image of the patterned surface was taken, followed by macrophage image, and the two images were merged.

Cell spreading area analysis

Cells were photographed and evaluated for cell spreading area using bright field microscopy, using a Nikon Eclipse TE300 inverted fluorescent microscope. The data shown represent means and standard error of the mean (SEM) of three experiments and six fields. The number of cells analyzed was from 110 to 345 cells for each data point. Cell spreading area was analyzed using Image J software, downloadable from the National Institutes of Health. Statistical tests were performed using analysis of variance (ANOVA) with p=0.05.

Results and discussion

Polysaccharide patterning and characterization

Prior to surface patterning, uniform homogenous surfaces of dextran, hyaluronic acid and APTES were made on silicon wafers and glass to verify the surface reaction. Surfaces were characterized by contact angle and ellipsometer as described in previous publications31. Briefly, silicon oxide surfaces are chemically modified to contain 3-aminopropyl triethoxysilane (APTES), and subsequently reacted with oxidized dextran or hyaluronic acid. The contact angles of APTES-, dextran- and hyaluronic acid-modified surface are 55 ± 6°, 35 ± 2° and 21 ± 5°, respectively. On the silicon oxide surface, the average thicknesses of the APTES-, dextran- and hyaluronic acid-modified surface are 10 ± 1 Å, 9 ± 3 Å and 18 ± 4 Å, respectively. These data are consistent with previous results and other published values32.

The photodegradation and microcontact printing methods were successfully used to pattern rhodamine-dextran, fluorescein-hyaluronic acid and their combination on a surface. Figure 1 shows the scheme for the fabrication process. The fluorescence microscopy images, Figure 2A, 2B and 2C, demonstrate the success of the patterning for rhodamine-dextran/glass, fluorescein-hyaluronic acid/glass, and rhodamine-dextran/fluorescein-hyaluronic acid, respectively. Both methods do not require extensive equipment and can be carried out in biological laboratories. This is especially true using the photodegradation method since the masks can be purchased readily. Although both techniques can yield the same patterned sizes, the surface chemistries for the patterns are different. While the microcontact printing method results in rhodamine-dextran alternating with APTES-glass (rhodamine-dex/APTES-glass), Figure 2A, the pattern generated by photodegradation is a combination of rhodamine-dextran and glass. This is important because the APTES-glass is hydrophobic and glass is not. Similarly, the hyaluronic acid patterning results in fluorescein-hyaluronic acid/APTES-glass for microcontact printing, Figure 2B.

Figure 2.

Figure 2

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Fluorescence images of three different patterns using microcontact printing, namely, (A) rhodamine-dextran/glass, (B) fluorescein-hyaluronic acid/glass and (C) rhodamine-dextran/fluorescein-hyaluronic acid features. Scale bar length is10 µm.

After preparing the rhodamine-dextran/glass pattern, a second polysaccharide can be attached to the glass regions using the photodegradation method or the APTES-glass region using microcontact printing. In our study, we chose hyaluronic acid because it is native to human tissues as well as a constituent of the endothelial glycocalyx surface. Hyaluronic acid was tagged with fluorescein which emits green. The binary pattern of rhodamine-dextran and fluorescein-hyaluronic acid is shown in Figure 2C using microcontact printing. The rhodamine-dextran/fluorescein-hyaluronic acid patterned surface can also be made using the photodegradation method. Figure 3A is an image of the rhodamine-dextran and Figure 3B is the fluorescein-hyaluronic acid. Merging the two images, we demonstrate that both polysaccharides form distinct regions corresponding to either rhodamine-dextran or fluorescein-hyaluronic acid.

Figure 3.

Figure 3

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Binary polysaccharide patterns of rhodamine-dextran (perimeter region)/fluorescein-hyaluronic acid (square core regions) prepared by the photodegradation method. (A) Fluorescence image of rhodamine-dextran using 535–585 nm excitation and 600–655 nm emission filters, (B) fluorescein-hyluronic acid using 460–490 nm excitation and 515–550 nm emission filters and (C) the merged images from (A) and (B), showing both rhodamine-dextran and fluorescein-hyluronic acid on one single patterned substrate.

In addition to fluorescence microscopy, the patterned surfaces made by microcontact printing were characterized using atomic force microscopy (AFM). Figure 4A and 4B are tapping mode AFM images of patterned surfaces of dextran/glass and hyaluronic acid under dry and wet conditions, respectively. For this analysis we measured the stripes because the AFM scanning size is limited to ~ 90 µm, which would give an incomplete view of the 90 µm square pattern used. AFM images reveal patches of dextran and hyaluronic acid across the surface coverage. The glass regions are relatively flat with roughness on the order of 1 nm, suggesting that polysaccharides do not attach to the glass. Consistent with the master pattern, the AFM images of the patterned surfaces show 10 µm periodicity with 5 µm width for the polysaccharide regions. The root mean square (RMS) values of the difference in the height irregularities were analyzed on the non-aggregate regions. The polysaccharide patches may have multiple polymer chains, and thus, these dextran or hyaluronic acid aggregates are not quantified. The RMS values of the Δheight are 2.0 ± 0.5 nm and 3.5 ± 0.2 nm for the dextran pattern on glass under dry and wet conditions, respectively, where Δheight is the difference between the height of the dextranized region and the height of the glass region. Similarly, for the hyaluronic acid pattern, the values of Δheight RMS are 2.8 ± 0.2 nm and 6.9 ± 0.7 nm for dry and wet conditions, respectively. This degree of swelling is similar to that found by Ferrer et al. in recent experiments in which the polysaccharides swelled by 2–3 fold, depending on the hydrogen bonding33.

Figure 4.

Figure 4

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AFM topography images of (A) rhodamine-dextran/glass and (B) fluorescein-hyaluronic acid/glass pattern taken dry and in liquid, tapping mode. The periodicity is 10 µm with 5 µm width for the polysaccharide regions. The dextran pattern shows patchy areas of dextran clusters, whereas the hyaluronic acid pattern has more uniform coating on the surface. (C) Root mean square (RMS) values of the height difference between the dextranized area and the glass. Scale bar length is 10 µm.

Both microcontact printing and photodegradation can yield micrometer-sized patterned surfaces. However, we found the photodegradation method to produce more consistent edges and borders. To prepare reproducible patterned surfaces using microcontact printing, requires much trial and error. Moreover, microcontact printing requires a master pattern on a silicon wafer which may not be easy to obtain. For these reasons, the photodegradation method was used to create patterns to study human macrophage adhesion.

Human macrophage adhesion on the homogenous surfaces

Macrophage spreading area was analyzed for each uniformly modified surface. Figure 5A–E presents representative images of macrophage morphology on surfaces of APTES, glass, culture dish, dextran and hyaluronic acid, respectively. On all the surfaces studied except hyaluronic acid, macrophages adhered and spread well, demonstrating distinct lamellipodial extensions. There were also subpopulations of macrophages on these surfaces which attached, but which did not spread significantly. The spreading cells took on elongated, polygonal or circular morphologies. On the hyaluronic acid surfaces, the adherent cells remained much smaller in their attachment and spread coverage area in comparison to the cells on the other surfaces. The macrophages on the hyaluronic acid-modified surfaces primarily remained rounded, and the cells also formed multi-cell clusters, unlike the cells on the other surfaces which remained spatially isolated from each other. The few cells that attached to the hyaluronic acid surface were lacking the spreading feature of large lamellipodi, but some cells did elongate in forming clusters with other cells.

Figure 5.

Figure 5

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Representative images of macrophage attachment and spreading on (A) APTES, (B) glass, (C) culture dish, (D) dextran and (E) hyaluronic acid for 3 days. (F) Normalized macrophage spreading area analysis on various homogenous surfaces. The macrophages attach and spread well on APTES, glass, culture dish and dextran, except the hyaluronic acid surface. The cells that attached on hyaluronic acid form clusters, showing preference for cell-cell adhesion rather than cell-surface adhesion. Scale bar length is 50 µm.

In Figure 5F, cell spreading area was plotted as a function of the different types of modified surfaces. Comparing spreading that occurred on the hyaluronic acid-modified surface to that of the other surfaces studied, cells spreading was statistically significantly less than on each of the other surfaces. This indicates that the hyaluronic acid-modified surface presents far less favorable chemistry for macrophage adhesion than do than dextran, glass, APTES-modified glass and culture dish surfaces. In addition, macrophages favored cell-to-cell interactions represented by formation of clusters of cells when adhering to hyaluronic acid, but not the other surface chemistries, as evidenced by the images in Figure 5A–E.

The macrophage spreading analysis also indicates that macrophages favor APTES-modified surfaces to dextranized or glass surfaces, with statistical differences present at 95% confidence levels using analysis of variance. Furthermore, comparing results for the two control surfaces, culture dish and glass, macrophages spread more on the culture dish than on the glass. That the hyaluronic acid-modified surface proved to be the least favorable for macrophage adhesion and spreading suggests that macrophage adhesion is favored by the cationic surfaces. This is further supported by the finding that the highest spreading occurred on the APTES-modified surface. Macrophage spread less on the anionic surface relative to the other surfaces in this study. This is consistent with studies by Brodbeck et al. who showed higher rates of apoptosis for macrophages adherent to anionic surfaces12,13.

Our results for cell clustering are also in close agreement with results of Gupta et al. showing that fibroblasts cultured on gels composed of hyaluronan and methylcellulose exhibit minimal cell attachment to the gel surface, with the majority of fibroblasts adhering to each other in clumps34. Additional studies have shown that mesenchymal stem cell spreading on hyaluronic acid hydrogels is directly proportional to the addition RGD peptides35. These results strongly suggest that hyaluronic acid by itself is a substrate that resists cellular attachment. Indeed our results presented here less macrophage attachment and spreading on hyaluronic surfaces than on the other materials tested.

While hyaluronic acid and dextran are both negatively charged polymers, the average macrophage spreading areas on these two surfaces are drastically different. This indicates that surface charge is not the only parameter dictating elements of the macrophage adhesion. Comparing the primary structure of the dextran and hyaluronic acid, both are found to be random coil36,37 in solution. Detailed studies of hyaluronic acid using NMR have shown that it does have a more complex structure than the original picture. Scott et al. showed that hyaluronic acid has extensive hydrogen bonding that forces the polymer to form a 2-fold antiparallel helix structure with hydrophobic domains38,39. These helices form a tertiary structure, consisting of 2-fold helices in two planes at right angles bonded by hydrophobic interactions and hydrogen bonding. This suggests that the shape of hyaluronic acid also plays an important role in governing macrophage adhesion. Further studies on the structure of hyaluronic acid immobilized on surfaces will provide additional information regarding how molecular structure modulates surface resistance to cellular adhesion.

Human macrophage adhesion is directed by surface micropatterning

In general, the cells did not align with any preferred orientation, but rather, they preferentially attached and spread on specific favorable surface chemistry. Studies have shown that cells can orient according to surface chemistry40–43. However, since the length scale of our surface chemical heterogeneity is on the micrometer length scale, a size scale which is comparable to, or larger than, the cells, specific cell orientation is not expected to occur.

On rhodamine-dextran/glass patterned surface, the cells did not preferentially attach or spread on the 90 µm square pattern or the 22 µm stripes, as is shown in Figures 6A and 7A. This is expected and is consistent with the results of the spreading area analysis on the homogeneous surfaces. As shown in Figure 5F, the spreading areas are not statistically significantly different on the dextranized and glass surfaces. The macrophage adhesion on rhodamine-dextran/glass pattern is in agreement with the adhesion on the homogenous surfaces.

Fig. 6.

Fig. 6

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Normalized macrophage spreading area analysis on various homogenous surfaces. Each bar represents the mean spread area of 110–345 cells of six fields in three experiments. Error bars indicate standard error of the mean (SEM). Statistical significance is for p < 0.05 using analysis of variance. Symbols (#), (*), (**) and (***) indicate statistically significant deviations from hyaluronic acid surface, dextranized surfaces, APTES surface and glass, respectively.

Figure 7.

Figure 7

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Representative images of macrophage attachment and spreading on 90 µm square for (A) rhodamine-dextran/glass, (B) fluorescein-hyaluronic acid/glass and (C) rhodamine-dextran/hyaluronic acid patterns using photodegradation method for 3 days. On the rhodamine-dextran pattern on glass, the macrophages have no preferential attachment or spreading. On the other hand, macrophages on the fluorescein-hyaluronic acid pattern on glass tend to attach and spread on the glass regions and only few cells are on the hyaluronic acid regions. The macrophages on the rhodamine-dextran and hyaluronic acid pattern show pronounced preferential attachment and spreading on the dextranized regions. Scale bar length is 50 µm.

The fluorescein-hyaluronic acid pattern on glass shows some preferential attachment and spreading on the glass regions for the 90 µm square pattern and the 22 µm stripes, as is evident in Figures 6B and 7B. The most dramatic preference is demonstrated by the rhodamine-dextran/fluorescein-hyaluronic acid 90 µm square patterned surface, which distinctly displays the success of using surface patterning to direct site-specific cell adhesion (Figure 6C). In this binary polysaccharide pattern, most of the macrophages preferentially attach and spread onto the rhodamine-dextran regions and avoid adhering within the fluorescein-hyluronic acid-modified area. This is similarly observed for the 22 µm stripes as shown in Figure 7C. However, the preference is not as pronounce as the 90 µm square pattern because the width of the grid is 35 µm. It appears that the 22 µm width on the stripes is smaller than the size scale of the macrophages. Our observation of more favorable cell attachment and increased spreading on the patterned surface of hyaluronic acid/glass and the rhodamine-dextran/hyaluronic acid is consistent with several cell studies on patterned surfaces involving cell lines such as fibroblasts44, epithelial cells45,46, endothelial cells47,48 and macrophages11,49. This surface-directed cellular adhesion can serve as an excellent method to control inflammatory cell behavior for applications in tissue engineering, biomedical device development as well as diagnostic procedures and therapeutic interventions.

Conclusions

Microstamping and photodegradation methods were used successfully to create patterned surfaces bearing two polysaccharides, dextran and hyaluronic acid, which were then characterized and examined further in a biological model of inflammatory cell adhesion. Human macrophage THP-1 adhesion on patterned surfaces of dextran/glass, hyaluronic acid/glass and dextran/hyaluronic acid was evaluated. Hyaluronic acid-modified surfaces resisted macrophage adhesion relative to other surfaces studied, including the culture dish, glass, APTES and dextran. On the dextran/hyaluronic acid patterns, macrophages are surface-directed to the dextranized regions. Both surface chemistry and geometry can limit cell interaction with the coated region of the surface that more greatly favor cell adhesion. This is evidenced by the cell spreading area and the cell morphology. These findings are relevant to the fundamental understanding of cell–substrate interactions and development of the surfaces for anti-inflammatory response in biomedical applications.

Figure 8.

Figure 8

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Representative images of macrophage attachment and spreading on 22 µm stripes for (A) rhodamine-dextran/glass, (B) fluorescein-hyaluronic acid/glass and (C) rhodaminedextran/hyaluronic acid patterns using photodegradation method for 3 days. Similar to the square pattern, the macrophages on the 22 µm stripes of rhodamine-dextran pattern show no preferential attachment or spreading. On the fluorescein-hyaluronic acid pattern on glass or rhodamine-dextran and hyaluronic acid pattern, majority of the macrophages attach and spread on or close to the glass or dextranized regions, respectively. The 22 µm size appears to be smaller than the size of the macrophages, and thus, cells extend to the unfavorable regions of hyaluronic acid. Scale bar length is 50 µm.

Acknowledgements

This work was supported by NIH grants R01-HL060230 and T32-GM007612. RJC acknowledges partial support from NSF/NSEC (DMR08-32802) and NSF/Polymer Program (DMR09-07493). Use of University of Pennsylvania Nano/Bio Interface Center AFM/TIRF instrument is acknowledged.

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