The Role of Sensor Surface Chemistry Topography and Energetics on Promoting Cell Recognition and Function

The surface properties of sensors that cells and tissues recognize (through initial protein interactions) in vivo or in vitro are chemistry, topography, and energy. As surface chemistry and topography both contribute to surface energy, which is directly

CeU/ttesue-imptanL interface

Topography & Chemistry

Roughness

Rrms:root mean square roughness: y5V: interfactal tension between so!id and vapor; ysl : interfacial tension between solid and liquid; yLV: interfacial tension between liquid and vapor RGDiarginine-glycine-aspartic acid

Fig. 3 Interactions between cells and sensor surface properties at the implant-cell interface related to surface wettability, many researchers have been using surface energy to characterize the interface between materials and cells. Figure 3 summarizes the sensor (or implant)-cell interface and interactions between cells, proteins, and sensor surface properties. It is worth remembering that proteins always dictate the interactions between surface properties and cells in an aqueous environment (body fluids, culture media, etc.). Therefore, understanding the impact of surface properties on protein adsorption and activity can also be an effective tool to explore biological responses on different sensor surfaces. In this section, we follow these rationales to explain the roles surface chemistry, topography, and energetics play on cellular functions, emphasizing how nanotechnology is promoting such interactions.

3.3.1 Surface Chemistry

There are many factors of surface chemistry that affect biological responses on sensors, such as the inherent chemistry (i.e., the chemical or phase composition and crystallinity of materials), possibly conjugated functional groups/molecules on the surface, and surface charge/polarity due to the configuration of such chemical groups. Different types of implant materials have been shown to be either bio-inert or bioactive, which results in either morphological fixation or bioactive fixation to the host tissue (Cao and Hench 1996). The different states of fixation indicate that the inherent chemistry of materials alters the responses of biological systems, and this information has guided researchers to seek a variety of implant materials. For example, the materials for orthopedic implants range from metals (CoCrMo alloys, titanium (Ti) and its alloys, and stainless steel), ceramics (alumina, zirconia, titania, and hydroxyapatite), ultra high molecular weight polymers (polyethylene, polyurethane, and poly-lactic-co-glycolic acid [PLGA]) to biologically

CeU/ttesue-imptanL interface

Topography & Chemistry

Roughness synthesized substances (such as mineralized complexes of collagens, calcium, and phosphate) (Balasundaram 2007).

On the other hand, modifying surfaces with functional groups have demonstrated a crucial role in influencing cell responses regardless of the underlying inherent chemistry. For instance, a wide range of peptides (most notably, arginine-glycine-aspartic acid (RGD)) have been conjugated on various material surfaces (Ti and its alloys, hydrogels, polymers, etc.) to improve bone cell functions in vitro and in vivo (Schuler et al. 2006; Kroese-Deutman et al. 2005; Picart et al. 2005). In addition, it is also believed that electrostatic interactions play a role in biological responses to implant materials since cell membranes carry charges and virtually all interfaces are charged in aqueous solutions (Haynes and Norde 1994; Wilson et al. 2005). An observation conducted by Qiu et al. (1998) revealed that positively charged indium tin oxide enhanced the adhesion of rat marrow stromal cells but impaired subsequent cell spreading and differentiation. Itoh et al. (2006) created electrically polarized hydroxyapatite with pores and reported increased bone growth and decreased osteoclast activity. They attributed these effects to the electrical polarity on surfaces with pores.

Clearly, one aspect of how nanotechnology has been used to improve bone growth has been through altered surface chemistry. Nanotechnology exhibits a great potential to improve implant efficacy by manipulating the discrete surface chemistry regions of nanomaterials. There are several reasons for this:

1. Nanomaterials can provide much larger surface areas, more substructures (such as grain boundaries), and more active surfaces for chemical modification. For instance, nano-fibrous poly(L-lactic acid) scaffolds modified by entrapping a large amount of gelatin molecules on the scaffold surface demonstrated improvements in osteoblast adhesion, proliferation, and compressive modulus (Liu et al. 2006).

2. A variety of novel nanomaterial shapes (dots, rods, tubes, cages, etc.) can be used for patterned, anisotropic, or multiple functionalizations. One example is that incorporating segments of growth factors and antibiotics into anodized nanotubular Ti can further enhance new bone formation and inhibit infection (Yao and Webster 2006).

3. Controllable assembly of nanomaterials can be realized or adjusted to certain chemical effects by a bottom-up strategy that establishes structures from tiny building blocks (e.g., atoms, molecules, etc.). For example, layer-by-layer nano-assembly of poly(lysine)/alginate above a gelatin (or extracellular matrix) surface resulted in a 200-fold decrease in the adhesion of human fibroblasts (cells that contribute to soft, not hard, granulation tissue formation) compared to the untreated surfaces (Ai et al. 2003).

3.3.2 Topography and Roughness

Another predominant surface property that can be easily modified through nano-technology to build better interfaces between sensors and bone is surface topography.

Among several ways of describing surface topography, roughness is a major parameter; its effects on bone tissue/cell responses has been studied intensely during the last two decades (Thomas and Cook 1985). Biologically inspired by the hierarchical micron-to-nanostructure of bones, which osteoblasts are naturally accustomed to in the body, an approach of creating nanometer roughness or nano-scale features on orthopedic implant surfaces can increase the efficacy of many orthopedic implant chemistries. This rationale has been applied to a variety of nanoscale materials (including metals (Webster and Ejiofor 2004), carbon nano-fibers/nanotubes (Price et al. 2003), polymers (Washburn et al. 2004), ceramics (Webster et al. 2000), and polymer/ceramic composites (Webster and Smith 2005)), and increased osteoblast functions (such as adhesion, proliferation, differentiation, and calcium deposition, etc.) on these nanostructured materials have been reported. Meanwhile, a general positive correlation between nanoscale roughness and biological responses (i.e., increased roughness corresponds to enhanced osteoblast functions) has been established (Anselme et al. 2000; Linez-Bataillon et al. 2002). One explanation of the nanometer roughness-enhanced osteoblast functions is that large surface areas associated with increased roughness can adsorb more proteins (fibronectin, vitronectin, etc.) important for osteoblast adhesion. However, other studies suggested that proteins adsorb differentially with variations in nanoscale surface roughness (Wilson et al. 2005), perhaps relating nanometer surface features to changes in surface energetics important for controlling selective protein adsorption.

Several other nanoscale topographical features that cannot be described simply by roughness may also mediate bone cells responses at the bone-sensor interface. Texture and alignment of surface patterns have been reported to influence both osteoblast functions and orientation on materials. For example, Biggs et al. (2007) reported a decrease in osteoblast adhesion and spreading on highly ordered nanopits (120 nm in diameter and 100 nm in depth) than randomly distributed ones, noticing that surface roughness on both substrates was similar because the number of pits per area on each was approximately the same. Zhu et al. (2005) created nanoscale polystyrene grooves with spacings of 150 nm and a depth of 60-70 nm and observed alignment of osteoblast-like cells and cell-produced collagen fibers along nanogrooves. Recently, thickness gradients of coatings in the nanometer regime have also been found to influence bone cell responses. For instance, a poly(3-octylthiophene-2,5-diyl) film with thickness gradients (120-200 nm) showed a higher proliferation ratio compared to tissue culture polystyrene controls after 1 day of incubation, while cell adhesion after a period of 4 h was not affected by changes in thickness over 10-60 nm (Rincon et al. 2009).

3.3.3 Wettability and Surfaces Energetics

To further understand the effects of nanoscale surface topography and roughness on osteoblast responses, researchers have defined surface energy. Surface energy is closely related to wettability (hydrophobicity or hydrophilicity) of a surface, and the basic relationship is given by Young's equation:

cose = (Ysv -YSL)/YLV, where d is the contact angle, 7SV is the interfacial tension (or surface energy) between the solid and vapor, ySL is the interfacial tension between the solid and liquid, and yLV is the interfacial tension between the liquid and vapor.

It has been widely observed that hydrophilic surfaces are energetically favorable for the adhesion and subsequent activities of osteoblasts as well as many other types of cells, probably relating to the increased adsorption of several hydrophilic proteins than hydrophobic proteins less important for cell adhesion. Measuring surface energy demonstrates an association of the relatively high surface energy on hydrophilic surfaces compared to hydrophobic ones, and the criterion of ca. d = 65° that differentiates between the two regimes has been suggested (Vogler 1999; Lim et al. 2008). One clear study created both hydrophilic (high surface energy) and hydrophobic (low surface energy) surfaces by modifying chemistry on quartz surfaces and showed that hydrophilic surfaces induced homogeneously-spaced osteo-blastic cell growth and mineral deposition, and enhanced the quantity (e.g., area) and quality (e.g., mineral-to-matrix ratio) of mineralization by osteoblasts (Lim et al. 2008). The authors suggested that surface energy effects on osteoblast differentiation, especially mineralization, could be correlated with surface energy dependent changes in spatial cell growth. Roughness also alters surface energy and resultant wettability, which establishes critical connections between topography and surface energetics. Although it has not been well substantiated, a trend of increased surface energy resulting from the increased presence of nanometer surface features enhances osteoblast functions has been shown in a number of studies (Anselme 2000; Das et al. 2007; Khang et al. 2008).

In summary, surface energy is closely related to surface wettability and can be modified through both surface chemistry and roughness. An idea of integrating surface chemistry, topography (roughness), and wettability into a unified expression of surface energetics and correlating such properties with biological responses will be of great importance to the understanding and mediation of protein, cell, and tissue responses on nanotechnology-created sensor surfaces.

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