bk-2012-1120.ch015

Chapter 15

Downloaded by UNIV QUEENSLAND on June 15, 2013 | http://pubs.acs.org Publication Date (Web): December 12, 2012 | doi: 10.1021/bk-2012-1120.ch015

Effect of Microtopography on Fibrocyte Responses and Fibrotic Tissue Reactions at the Interface David W. Baker and Liping Tang* Bioengineering Department, University of Texas at Arlington, Arlington, Texas 76019-0138 *E-mail: [email protected]

Governing the biomaterial mediated tissue response is critically importance for the future design and improvement of many medical implants. As such, an enormous amount of research effort has been placed on seeking a means to alter or control implant associated tissue reactions. Ultimately, the numerous strategies attempted boil down to a single notion of manipulating proteins and cells at the material interface. While the mechanisms and processes governing such reactions are not entirely understood, surface modification techniques have become the primary strategy, encompassing a wide variety of methods to improve the biomaterial interaction. While methods such as surface chemistry, functionality, and hydophobicity have all lead to various degrees of improvement, surface topography may be the largest contributor to combat adverse tissue and cellular reactions. We focus our discussion on the recent advances of surface topography manipulation and its effect on specific cellular responses both in vitro and in vivo. In particular we and others have found that micropillar topography has a profound effect on cellular morphology, migration, differentiation, and expression in vitro. The resultant influence of micropillar arrays on tissue responses in vivo however have only just begun to be investigated. Surprisingly, our own research has revealed that fibrocytes, circulating fibroblasts, but not macrophages are mostly responsible

© 2012 American Chemical Society In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


for micropillar-mediated tissue responses in vivo. A better understanding of the interactions between fibrocytes and micropillar topography may provide critical information for improving implant safety and function.

With continued advances in biomedical devices including novel polymers and intricate fabrication methods for advanced material implants there is a demand, now more than ever, to control the foreign body response and the biocompatibility of implants. Unfortunately, almost all biomedical implants are plagued with significant adverse reactions with localized tissue contact. Implant associated inflammation and tissue fibrosis lead to implant encapsulation and infection often resulting in the rejection of many medical devices (1, 2). Poor tissue integration and the formation of thick fibrotic capsules surrounding implants contribute to the failure of many medical devices including, breast implants (3), drug delivery vehicles (4), and biosensors (5), as well as spine/joint (6, 7), and eye implants (8). To improve the safety and efficacy of medical implants, substantial research efforts have been placed on the development of novel strategies for altering implant-associated tissue reactions. An account of the foreign body reaction has been well established, while specific relationships between the various processes are not entirely understood. Most medical implants are covered with a layer of plasma proteins, seconds to minutes, following exposure to bodily fluids or blood (9). A few hours and days later, implants are surrounded by a large number of immune cells and a small number of fibroblast-like cells. Subsequently, collagen-rich fibrotic capsules are formed isolating the medical devices from the surrounding host tissue. After biomaterial implants are enclosed by adsorbed plasma proteins, a dense population of macrophages/monocytes forms prior to fibrotic tissue formation. It is therefore generally believed that biomaterial:protein interactions dictate biomaterial tissue compatibility and that biomaterial-mediated phagocyte interactions are responsible for the subsequent fibrotic tissue formation.

Surface Modifications on Cellular Responses Intensive research efforts have lead to a deeper understanding of protein:biomaterial and protein:cellular interactions. The results of these works have also lead to a plethora of surface modification techniques. These studies have uncovered that surface chemistry, roughness, charge, hydrophobicity, or even simple micro and nano topographical cues alter protein and cellular reactions in vitro. The question remains however as to how these surface modifications will hold up to the foreign body response in vivo. Adding surface functionality to a material has become an important modification technique to control protein and cellular interactions. By increasing surface functionality, it is possible to control the hydrophilicity, the interfacial free 340 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


energy, and/or the ionic nature of the surface properties while leaving the bulk material properties unaffected (10). Some of the more common functional groups imparted to biomaterials are carboxyl (-COOH), hydroxyl (-OH), methyl (-CH3), and amine (-NH2). While these functionalities have shown significant influences on protein interactions and alteration of cellular function in vitro, the influence of surface functionality on implant-mediated fibrotic tissue responses in vivo was insignificant (11, 12). The disappointing results may be influenced by the fact that the functional groups can only interact with cells at the interface. The effect of the surface functional groups may be substantially enhanced by increasing functionalized surface areas. To test this hypothesis, surface fuctionalization of polypropylene microspheres with varying species and densities of functional groups were tested for their ability to prompt fibrotic tissue responses (11, 13). Indeed, our results have revealed that microparticles carrying various functional groups prompt an altered extent of fibrotic tissue responses, including capsule thickness, collagen deposition, and cell infiltration around the implant (11, 13). Despite of these exciting observations, the effect of surface functional groups on tissue responses may be, in part, surface topography dependent. For example, it has been shown that substrates bearing different surface chemistry but similar topography prompt similar inflammatory responses, indicating topography as a major contributor to tissue repair (14). Therefore, several studies were carried out to investigate the influence of implant topography on tissue responses.

Implant Topography on Cellular and Tissue Responses The geometrical configuration of the topographical features has become a significant target in the design of biomaterials. Subjects such as porosity, pore size, patterning, alignment, interconnectivity, surface roughness and curvature have all become increasingly hot topics with the design of scaffolds and novel biomaterials utilizing pores, pits, channels, grooves, pegs, and pillars. These architectural changes, even on substrates of the same chemical makeup, have revealed significant differences in cell responses and protein adhesion in vitro. One example is that the notion of cell contact guidance, a cells ability to respond to surface features, has been proven critical in manipulating cellular behavior (15). Cells respond to environmental cues by adjusting their morphology, orientation, expression, and even characteristics through differentiation. As such, a wide range of topographical signals have been investigated in an attempt to control cellular functions. A simple modification is to enhance the surface roughness characteristics. Although random, not organized or confined, surface roughness has shown improvements in cellular attachment and expression over smooth surfaces (16, 17). Furthermore, surface porosity has been found to affect tissue vascularity. By analyzing tissue vascularization at the tissue interface, an early study found that larger pore polytetrafluoroethylene (PTFE) membranes (0.8-8 micrometer pore size) prompted 80-100 fold more vascular structure than small pore membranes (0.02 micrometer pore size) (18). Furthermore, for 3D scaffold constructs, vascularization has been shown to be significantly faster for pores with 341 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


a size greater than 250 µm (19). It has been documented by many studies that interconnected pores and high porosity of scaffolds maximizes cell penetration during seeding as well as nutrient supply and waste transport (20). Interconnected pores however are most often random. In the design of specific constructs, such as blood vessels, it may be beneficial to have higher degree of structural orientation (21). In a study on porosity characteristics of β-tricalcium phosphate it was found that the size of the interconnections between pores was more important for the degree of vascularization than the actual pore size (22). Interestingly, with similar scaffolds, it was found that the degree of fibrous tissue in-growth increased with decreasing pore size from 700 to 300 µm (23). The degree of organization and pore shape has also been studied for collagen formation. In vitro, large scale (200 µm) rectangular pores or diamond shaped pores were found to guide cell and collagen orientation more effectively than square shaped pores (24). While it is clear that the characteristics of porosity influence cell infiltration and vascularity, it has also been shown that protein adsorption, cell migration and cell differentiation are influenced not only by pore size but also by the pore curvature, suggesting an intimate mechanosensing cellular mechanism (25). Despite of these exciting observations, the mechanism(s) governing such topography-mediated tissue responses are mostly undetermined due to the fact that the substrates and characteristics vary significantly, and in earlier works parameters such as interconnectivity were not controlled. To identify the physical factors critical for differential cellular responses, surfaces with more sophisticated and controlled geometrical patterning are needed. Substrates with different sizes (from nanometers to micrometers) of channels, grooves and pillars have become available in recent years with the development of lithography and nanotechnology. The effect of these man-made structures on protein and cellular behavior has been widely studied. Surface features as small as 10 nm have generally been recognized to affect both protein adsorption and cellular responses (17). Similar to native cues of the extracellular matrix (ECM), nanotopography is theorized to provide biomimetic cell–modulating signals altering cell attachment, migration, and proliferation for cells such as osteoblasts, fibroblasts, endothelial, epithelial and macrophages (17). Along these lines micro-surface roughness has been shown to substantially enhance the mechanosensing of osteoblasts and increase bone formation (26). A study without osteoinductive media similarly found that osteogenic capacity can be tuned by controlling the diameter of mixed micro/nanoscale pits on polycaprolacton (27). This study found that topography alone was sufficient to promote in vitro bone formation by human osteoblasts. Alternatively, macrophage behavior may be modulated by nanogrooves in vivo altering cytokine secretion and ultimately affecting the foreign body response (14). Channels or grooves have also shown a remarkable ability to organize alignment and migration of cells in vitro. Endothelial cells for instance have been shown to preferentially align in the direction of the channels when coated with fibronectin (28). Tenocytes are similarly affected by microgrooves in the 50µm range impacting shape, alignment, and matrix organization (29). It has been further discussed by McNamara et al. that mechano-transduction from topography alignment cues (microgrooved silica glass 12.5µm wide 2µm deep) leads to chromosomal repositioning and gene regulation (30). This is thought 342 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

to be a result of tensile and compressive forces acting on the nuclear lamina (30). These studies and others clearly demonstrate that geometrical constraints such as patterning, space, height, and curvature, may dictate cellular attachment proliferation and even gene expression of many cell types.


Surface Topography on Implant Tissue Compatibility Channels and grooves have mostly been limited to in vitro studies with few deviations toward the in vivo setting. A murine study utilizing poly(e-caprolactone), poly(lactic acid) and poly(dimethyl siloxane) gratings from 250nm to 2µm found that macrophage behavior was affected in vivo primarily with cell adhesion on the larger sized gratings (14). In contrast however a study utilizing polystyrene disks with microgrooves from 1-10µm found no discernible differences in the tissue reaction between smooth and microgrooved implants in a goat model (31). Despite these and other differences, microgrooved substrates have made a significant impact on our understanding of cellular attachment and alignment cues in vitro, although alignment is primarily limited to a unilateral direction. Another disadvantage to microgrooves is the ability to alter the substrate rigidity, which more closely resembles the bulk properties of the material and is less precisely controlled with long unidirectional channels. To further investigate cellular attachment and alignment but also improve on the rigid flexible nature of the surface we turn to micropillars. Micropillars or pegs have several unique features that provide a good model implant for cellular studies. First the geometrical configuration of micro pillars including diameter, spacing and height are easily controlled by lithography or mold inversion process techniques. The pattern, typically square, hexagonal (Figure 1), or semiordered may also be specifically designed, and has been shown to either hamper (square orientation with small spacing) or favor (hexagonal orientation with larger spacing) cellular migratory responses. The shape of the pillar, curvature, or edge constraints, can also be concisely constructed (32). Micropillars additionally offer a unique flexibility or elasticity to surfaces that otherwise would be highly rigid. In conjunction, the surface area may be greatly increased and the hydrophilicity/ hydrophobicity may be altered on an intrinsically nonadhesive surface by the addition of micropillars (33). This unique characteristic is controlled by the density and stiffness of the pillars, which are ultimately effected by their spacing, height, and diameter. The stiffness of the pillars and the ability of the cells to exert contractile forces may have a significant influence on the cellular biology. Studies have shown that culturing cells on micropillars tends to promote cell attachment while reducing proliferation (34). By providing contact guidance, micropillars are observed to support cell adhesion and elongation as well as cytoskeleton orientation. In addition several studies have recently reported mechanical regulation of cell function or differentiation due to elastomeric changes based on pillar height (35).

343 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 1. Hexagonal arrangement of micropillars from (A) the top or (B) the side view. In both A and B the pillars are spaced 20 µm apart on center, are 25 µm in height, and have a diameter of 10 µm. (Adapted with permission from reference (36). Copyright 2011 American Chemical Society.)

Concise topographical changes by the alteration of pillar geometry and size characteristics can also result in a significant shift in inflammation and fibrosis around an implant. We have recently observed a drastic change in cellular responses to varying pillar geometries. Surprisingly we have found that different types of cells prompt varying responses to the same micropillar substrates (36). We focused our study on macrophages and fibroblasts as they are thought to be the main cells responsible for inflammatory and fibrotic responses. What we found is that differences in the height of the micropillars (14-25 µm) altered the attachment and growth of fibroblasts, increasing with height, however differences in the spacing of the pillars (20-70 µm) hampered growth of macrophages in vitro. The difference in fibroblast growth was in agreement with several previous studies which attribute the increased spatial surface area to greater extracellular matrix production and cell activation (33, 37). Interestingly the macrophage response was unexpected and contradictory to the general assumption that macrophages are solely responsible for triggering fibrotic tissue reactions. This indicated that the pillar topography may have had a greater impact on the fibroblast activation/ adhesion in vivo. Our in vivo results uncover significant differences in the tissue response at the biomaterial interface in cell density, capsule thickness, collagen percentage, granulation tissue thickness and angiogenesis (36). It is not clear however what the source of the fibrotic cells is, how surface topography affected the fibroblast response, and if this change alone can account for the differences in the tissue response at the interface. Coincidentally, recent studies have uncovered novel circulating fibroblast-like cells termed fibrocytes (38, 39). Fibrocytes, often denoted by either CD45+ or CD34+ in conjunction with either collagen I+ or vimentin+, have been shown in many recent works to play a pivotal role in the pathogenesis of pulmonary, renal, hepatic and dermal fibrosis (40–42). In wound healing these fibroblast-like precursor cells differentiate into myofibroblasts and secrete collagen, vimentin, actin, and other proteins which influence the developing fibrotic matrix. Our studies have revealed the presence of fibrocyte cells at the implant interface (36, 43). Interestingly it has been demonstrated in pulmonary fibrosis that fibrocyte recruitment corresponds directly with collagen production in the lung (44). In our investigation we similarly demonstrate that the fibrocyte response to the implant 344 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


correlates with the collagen production at the interface and the progression of the fibrotic reaction out to 14 days. We observe a maximal accumulation of fibrocytes around 10 days post implantation, followed by a decrease in numbers as the fibrocytes down-regulate expression of leukocyte CD34 and CD45 markers and up-regulate α-SMA during the transition to myofibroblasts (43). Myofibroblasts are found at the site of tissue injury and are believed to be critically involved in the healing process by secreting ECM proteins and promoting contraction (45). Several environmental factors such as the presence of transforming growth factor beta (TGF-β) have been shown to transition fibrocytes into myofibroblasts in vitro (46). Interestingly it has also been shown that other factors such as interleukin 1-beta (IL-1β) may function to maintain fibrocytes in a pro-inflammatory state leading to an increase in the inflammatory cell population during wound healing (47). We hypothesize that the microenvironment cues are altered by the various pillar geometries and cellular interactions, allowing fibrocytes and fibroblasts to have a more dominant influence on the tissue response. The question remains however as to how a difference in pillar geometry alters the micro-environmental cues.

Effect of Surface Topography on Protein Adsorption and Cellular Behavior at the Interface Several studies have suggested that micropillar features provide more surface area for cells to form focal connections (33, 37). Alternatively, it has been shown that patterning of polydimethylsiloxane (PDMS) surfaces with dot-like protrusions increased fibrinogen adsorption by 46% compared with the flat surface, while surface area was only increased by 8% (48). This alteration in fibrinogen adsorption was shown to influence fibroblast adhesion and morphology. Additionally it was found that human fibroblasts attempt to endocytose or internalize polymethylmethacrylate nanocolumns (160nm in height, 230nm spacing, and 100nm in diameter), showing a more macrophage-like morphology (49, 50). These differences show the vast potential of micropillars and controlled topographical features to alter cellular behavior. Altering the features of the micropillars such as height, spacing, diameter, and configuration also changes material substrate properties such as elasticity. It has been shown in several studies that cells are highly responsive to substrate stiffness or rigidity (35, 51). As an example, micropost rigidity has been found to impact stem cell morphology, focal adhesions, and cytoskeleton contractility leading to alterations in stem cell fate, or differentiation (35). We hypothesize that these mechanisms may similarly impact precursor fibrocytes in the foreign body response. Enhancing the predisposition of fibrocytes to differentiate into myofibroblasts leads to further collagen production and may also have a significant impact on the orientation and alignment of the collagen within the fibrotic capsule. As previously mentioned we have observed differences with macrophage and fibroblast cells in contact with smooth or pillared surfaces. These potential differences are demonstrated in the schematic shown in Figure 2 representing a hypothetical in vitro response. Shown here as fibroblastic 345 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


or phagocytic cells, we have observed significant differences from the initial interaction to the preferential interaction and accumulation of cells on the smooth and pillared substrates. While phagocytic cells preferentially adhere and spread on the smooth surface, fibroblastic cells become more prominent on the micropillar substrate. Fibrocytes may behave similarly with an enhanced response to the cues provided by micropillars. We hypothesize that this cell: surface interaction would increase fibrocyte signaling while decreasing phagocytic cell reactions. The difference in the resultant response may be more inflammatory on the smooth surface with an increase in factors such as IL-1β and TNF-α, or more wound healing with increased TGF-β and myofibroblast production.

Figure 2. Schematic of cellular response to smooth (A) and micropillar (B) substrates. After the initial cellular-substrate interaction, there is a preferential interaction of fibroblastic cells on the pillared substrate, and phagocytic cells on the smooth substrate. The resultant response shows a spreading cell morphology for both phagocytic (i) and fibroblastic (ii) cells on the smooth substrate. Alternatively the resultant response on the pillared substrate shows small low proliferative phagocytic cells (iii), and spindle shaped highly proliferative fibroblastic cells (iv). (All scale bars = 25 µm.) There are at least three main modes of cell mediation to the biomaterial surface, focal adhesions, close contacts, and extracellular contacts. Cells are guided by focal adhesions, which in turn are mostly altered by the initial protein adsorption to the implant. Interestingly the amount, composition and degree of host protein adsorption may be altered by specific topographical cues. This 346 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


simple shift in initial protein interaction with the surface has shown great propensity to influence the final cellular interactions and shift the inflammatory/ fibrotic response. This could either enhance or diminish the encapsulation effect which may result in enhanced functionality of the medical device. Micropillars have been shown to alter the protein attachment at the interface based on topography. An intrinsically hydrophilic material may lose its resistance to protein binding based on geometry (52). Alternatively, a hydrophobic material may lose its resistance to cell adhesion. This interaction may be primarily due to spatial changes in topography, however there are also intrinsic changes in the surface area and free energy. In addition it has been observed that there are significant differences in the micro and nano scale interactions. A recent study suggests that the adhesion and mechanical cues provided by the pillars alters the microenvironment, enhancing adhesive interactions with cells and the production of proteins that form mechano-chemical feedback (34). The alteration of these cues can have a profound influence on cellular behavior.

Figure 3. Schematic depicting theoretical protein adsorption, focal adhesion, and cell activation on smooth (A) and pillared (B) substrates, leading to the observed tissue reaction. Micropillar surfaces may provide increased protein adsorption, followed by greater focal connections, and altered cellular activation. A shifting orientation of cellular attachment and activation may lead to production differences and directional changes in matrix accumulation. Histology images are presented (hematoxylin and eosin staining) for subcutaneous PDMS film (smooth substrate) and pillar [20 µm spacing 25 µm height] implants at two weeks in a murine model. 347 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Shifting focal adhesions and directional organization of the cells on channels and grooves is found to have an additional impact on the later organization of the extracellular matrix. It is well known that channels or grooves alter the cellular morphology often in the direction of the grooves dependent upon geometrical constraints. In addition, it has been found that the primary organization of cells results in alignment of produced ECM and further orientation of subsequent cell layers in vitro, more closely resembling the native tissue (53). The ECM is made up of polysaccharides and proteins (54) and works to regulate cell function as both a positive and negative regulator of differentiation and gene expression (54). It has been observed that shifting cellular patterns show a resultant change in the ECM. This dramatic effect of ECM on cellular behavior has been described in several culture models such as hepatocytes, mammary epithelial cells, and keratinocytes (54). Based on the above analysis performed on channels we hypothesize that a similar mechanism would exist for the in vivo formation of the ECM in response to micropillars. Figure 3 outlines the theoretical difference in protein adsorption, cellular adhesion, and cell activation leading to the observed tissue reaction. Increased surface area and protein adsorption may lead to increased cellular adhesion on micropillar substrates over smooth surfaces (Figure 3). It is also possible that the increased focal contacts, as a result of changing pillar dimensions, would enhance the mechano-chemical feedback to the cell. Based on the geometrical constraints, or lack of constraints, the ECM formation would be laid down in a manner resembling the initial cellular attachment. Indeed, our histological analysis confirms similar trends for the smooth and micropillar surfaces (36). Therefore, with precise control of the micropillar parameters, such as stiffness or rigidity, it may be possible to engineer the orientation of the extracellular matrix.

Changing Collagen Alignment with Micropillar Topography The idea of contact guidance, cells orientation due to physiological topography is not new. The trends in research however are suggesting that there may be a greater impact on the protein arrangement due to topography which in turn alters the cellular response. With a shift in the cellular response the produced extracellular matrix may orient in a similar fashion as dictated by topography. It has been shown that cell expression, morphology, and even nucleus deformation are all responsive to the topography in vitro (55). On the other hand, matrix production and collagen alignment, while distinctly arranged by micro- and nanogrooves in vitro, may be responsive to micropillar substrates in vivo. As a model for topographical features, we investigated three spatial arrangements of micropillars (36). All pillars were 10µm in diameter in a hexagonal arrangement. To investigate the tissue response to various geometries, the interspaces and heights of the pillars were altered. Three spatial dimensions were investigated distancing the pillars from center to center at 20, 35, and 70 µm. Two pillar heights were investigated at 14, and 25 µm. We found that different cell types, specifically macrophages and fibroblasts, respond differently to the same microtopographical cues (36). In vivo we observe that higher micropillar 348 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


substrates gather more granulocytes and fibroblasts. In fact cellular density at the interface of taller pillar substrates increased more than 50% over the control. Additionally the capsule thickness was found to increase with pillar height but only becoming statistically significant when pillars were placed further apart. While the increase in cellular density may be closely related to a change in the tensile modulus or stiffness of the pillars, there is also a clear augmentation in the response resulting from the spacing or distance between pillars. Our results suggest that the greater distance and increased height had a substantial effect on the cellular activation and production of ECM. Taking a closer look at the tissue responses we similarly noticed that the granulation tissue (accumulation of phagocytic cells), neoangiogenesis, and collagen production where all enhanced by the increasing pillar dimensions.

Figure 4. Representative histological images and schematics of observed in vivo collagen alignment with PDMS micropillar samples. Collagen alignment is observed at the biomaterial interface with picro-sirius red staining taken with polarized microscopy. The birefringent images show collagen 1 fibril orientation for smooth (A), 20 (B), 35 (C), and 70 (D), µm spaced micropillars at a height of 25 µm. Schematics outline general orientation of collagen fibrils (black vectors) observed in the samples. Animal implantation study supports that the micropillar topography may shift the inflammatory fibrotic response dependent upon pillar height and spacing. The resultant change in vivo is a drastic difference in the amount of granulation tissue, which is important for wound healing, as well as the level of collagen production by resident and recruited fibroblasts. Interestingly investigation into specific collagen fiber alignment with polarized light microscopy demonstrates that the specific pillar orientation alters the layout of the collagen fibrils. Such 349 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


differential collagen fiber alignment can be observed in the birefringent images from picro-sirius red staining for collagen as well as representative vectors for the collagen fibril orientation (Figure 4). The smooth surface control and each pillar substrate, three pillar spacings and two pillar heights, were studied after two weeks in vivo. In the control sample (without pillar), implants are surrounded with dense collagen fibrils which are mostly oriented in parallel with the substrate or flow like a wave through the implant interface (Figure 4a). This response is also observed to be translated though the fibrotic capsule mimicking the same fiber directions when the capsule reaches the normal tissue of the epidermis. As the pillars become further spaced apart the fiber orientation begins to lose this intrinsic wave and become more segregated adapting to an un-parallel formation. In the 35 µm spacing arrangement (Figure 4c), there is clear organization at the interface of cells with the implant. In the 70 µm spacing (Figure 4d), the fibers seem disconnected with a greater percent at almost perpendicular angles to the pillar implant. This difference in organization may potentially have a profound influence on the fibrotic capsule contraction around the implant. It should be noted that similar trends were also observed for the 14 µm pillar height (not shown), but were less significant. It is clear that the micropillar configurations not only had a profound effect on the fibrotic outcome but also influenced the direction and orientation of the resultant ECM produced throughout the fibrotic capsule. This result may have further implications into the potential use for micropillar implants where it may be possible to orient the collagen fibers in applications such as anisotropic tissues for which collagen alignment is of utmost importance to the function of the tissue.

Implications/Applications Micro- and nanotopography has a high potential to alter cellular and protein interactions at the material interface. Our results suggest that surface topography affects fibrocyte responses and subsequent collagen production/orientation. By creating an un-parallel collagen matrix, the presence of surface micropillars at the interface may indirectly reduce fibrotic capsule contraction. Despite of many exciting findings, numerous gaps remain in our understanding on the influence of surface topography on cellular responses. For example; how surface topography affects cellular responses such as activation? How the microenvironment cues are altered by the various pillar geometries? What role increased protein adsorption and focal adhesions play? How pillar height and spacing affect fibrocyte responses? And why various cell types respond differently to the same topographical cues? The results of these works may have significant impact on the design of medical implants with improved safety and/or tissue reactivity. For example, many load-bearing soft tissues are subject to a high degree of mechanical anisotropy, including heart valves, blood vessels, tendons, skin, cartilage, myocardium and pericardium (24, 56). To develop physiologically equivalent replacements, the aim of tissue engineering is to mimic the native structure. It is believed that the basis for the anisotropy is the collagen fiber structure. While 350 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

there are several models that exist to study the nature of the collagen formation and alignment, the development of a substrate to direct cell function and matrix production in a physiologically equivalent manner remains a difficult challenge. For example, hydrogels are typically used to study collagen formation, however load constraints must constantly be applied to control the structural organization of the collagen fibers (56). Micropillars may offer an alternative approach in the design of anisotropic collagen fibril formation with concise design parameters to control the cells, matrix production, and alignment.


Acknowledgments This work was supported by NIH Grant EB007271 and AHA-South Central Affiliate Grant-in-Aid Award.

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