Biocompatibility – Cell Responses to Surface Chemistry

1] INTRODUCTION:

The surface of the biomaterial plays an instrumental role in the determination of biocompatibility, associated biological and interfacial responses that, in turn, influence and yield implant success. The surfaces of the biomaterial can be modified for manipulation of implant features by physicochemical, biological and processing methodologies of surface patterning and assemblage (Temenoff and Mikos, 2008, pp. 238-350). The reactions at the surfaces and interfaces of the implant can be biologically originated and initiated. Specific cellular responses can be provoked by the generation of perfect surfaces that facilitate ideal implant performance and paves way for direct regeneration of new tissue.
Innovative biomaterials should be capable of meeting the nutritional and biological needs of the host tissue to employ specific cell proliferation. A vague understanding of the detailed interface and surface interactions between biomaterials and the host tissues can lead to several clinical complications such as fibrosis, inflammation, foreign body responses etc. The initial cell responses depend on the chemical composition, surface energy, roughness, topography and surface chemistry (Temenoff and Mikos, 2008, pp. 238-350).

The cell receptors react with the specific proteins/other cells to form the major surface reactions determining physiological stability of the cell (Sevastinov, 2002, pp.20-30). Surface chemistry depends foremost on the signalling complexes of ECM that include the growth and differentiation factors of the cell, affecting cell proliferation and cell differentiation, determining the matrix remodelling of the tissue organs (Sevastinov, 2002, pp.20-30). Integrin mediated cellular activities contribute to the cellular adherence of ECMs via the actin filament connection of their cytoskeleton to the ECM (Sevastinov, 2002, pp.20-30).
Improvements in haemocompatibility and biocompatibility by irreversible absorption of a layer of albumin on biomedical implants have been exemplified by Sevastinov. The research conducted at the University of Utah and Texas prove that the haemocompatibility of polyurethane TAH and LVAD devices enhanced the generation of an irreversible layer of albumin by surface inclination increment (Sevastinov, 2002,pp.20-30). The detailed descriptions of surface chemistry-modification mechanisms, clinical relevance, medical device applications and associated cellular responses are described in the following sections.

2] CATEGORIES OF CELL-BIOMATERIAL INTERACTIONS AND MECHANISMS OF SURFACE CHEMISTRY MODIFICATIONS TO ELICIT SPECIFIC CELLULAR RESPONSES:

A material surface can be understood to be a division of planar defects. The surface atoms exhibit an unequal level of bonding with other atoms and an extra magnitude of energy is concomitant with the region due to void valence shells (Temenoff and Mikos, 2008, pp. 238-350). The excess energy, due to surface tension which forms a thermodynamically unstable state, adsorbs atoms/molecules to minimise surface tension (Temenoff and Mikos, 2008, pp. 238-350). Molecular adhesion to solid surface /adsorption occurs in physiological conditions on implant surfaces. Adsorbate on implant surfaces is composed of ions, water and proteins. The adsorbate is the coated surface to which the body reacts. The protein adsorption is favoured by the hydrophobicity of the surface and the charge of the surface. General trends exhibited by biomaterial surfaces show enhanced protein adsorption with an increase in hydrophobicity, though exact analysis can be yielded from contact angle analysis of the surface. The factors of steric repulsion and surface roughness, Ra also contribute to the phenomenon of protein adsorption (Sevastinov, 2002, pp.20-30).

The two major divisions of cell-biomaterial interactions are:
Ø Specific interactions – include the protein adsorption containing specific ligands. The integrin binding affinity of different functional groups can be an example. This is explained in detail in Section 3 (Sevastinov, 2002, pp.20-30).
Ø Unspecific interactions – it is difficult to control the kinetics and interactions of this kind. These depend on the properties common to the multiple cell types. A non-physiological interface exists which leads to impairment of cell activity. This depends on the cell surface properties, negative charge of the plasma membrane, and lipophilic proteins of the ECM which lead to mediation of the unspecific adhesion of biomaterials (Sevastinov, 2002, pp.20-30).
The different surface chemistry approaches utilised for modifying, manipulating and controlling cellular responses are modifications of:

A] The morphological nature – these include tissue ingrowth encouraging porous coatings (HAP coatings etc.), grooved surfaces to facilitate epithelial down growth and direct formation of bone specific to an implant region.

B] The chemical nature – these include (Sevastinov, 2002, pp.20-30):
· Glow discharge to increase surface-free energy and tissue adhesion.
· Cross-linking of polymeric surfaces to attain decrease in surface permeability, increase in Ra.
· Plasma treatment for the introduction of new functional groups.
· Ion implantation to improve corrosion and wear resistance.
· Macromolecule grafting such as PEO for reduction of protein adsorption, cell adhesion.
· Production of amino (positive) surfaces/ acidic or sulfonate surfaces.

C] Biological modification – biomolecule immobilisation of the biomaterial being used (Sevastinov, 2002, pp.20-30):
· RGD, heparin/heparin sulphate-bind peptides, proteins such as fibronectin or growth factors for enhancement of cell adhesion and growth.
· Immobilisation of phosphorycholine for resisting protein and cell adhesion for blood contact applications.

The State Centre for Blood Compatible Biomaterials at the Research Institute of Transplantology and Artificial Organs, Moscow, has related haemocompatibility/blood biocompatibility of an implant with hydrophilisation and hydrophobic behaviour with the aid of irreversible passivation with protein. Biological modifications of biomaterials modify the surface chemistry and other properties in the methods as given below:
ü Albumin aided/unaided surface hydrophobisation
ü Biologically active compound immobilisation aided/unaided surface hydrophilisation
ü Protein-aided surface passivation (uses albumin/plasma)
Research conducted by Sevastinov indicates that albumin layer adsorption during processing increased the inclination of the surface to the irreversible phenomenon of surface passivation. This was demonstrated in polyurethane devices of TAH and VAD. Preliminary passivation of the surface in conjunction with heparinisation/polymeric coating for the development of heparin affinity was found to increase the magnitude of surface-bound heparin and the reduction of the release of the platelet factor. This process was utilised by the State Centre for Blood Compatible Biomaterials at the Research Institute of Transplantology and Artificial Organs, Moscow, for the modification of vascular grafts, bioprostheses, heart valves and haemodialyser reprocessing.

Surface chemistry is capable of inducing putative changes in the focal adhesion and integrin binding in biomaterial surfaces. Self-assembled monolayers of alkanethiols on gold were found to induce changes in the osteoblastic proliferation, adhesion, spreading and differentiation at a differential rate when functionalised with OH, COOH, CH3, NH2 groups. Studies conducted by Keselowsky and colleagues indicate that SAMs of alkanethiols on gold with human plasma FN osteoblast cell line promoted a trend of OH> NH2=COOH>CH3 for α5-β1 binding (Keselowsky and Collard, 2004, pp.5947-5954). αv- β3 integrin binding followed a pattern of COOH>NH2>>OH=CH3. Focal adhesion assembly was found to alter the FAK signalling, cell-matrix adhesion composition and structure (Keselowsky and Collard, 2004, pp.5947-5954). Protein adsorption generally occurs at a significant level on a hydrophobic surface, but the system features and functionalisation of the surface and surface chemistry modification can alter the general trend.

Cell adhesion to biomaterial surfaces can be controlled by the immobilisation of short chain oligopeptides that specifically bind to protein domains. RGD, found in several different ECM proteins such as FN, VN, laminin and collagen, is capable of directing and controlling cell adhesion and bioactivity (Keselowsky and Collard, 2004, pp.5947-5954). Martin and co-workers immobilised osteopontin on pHEMA to promote endothelial cell adhesion. They found that immobilisation of osteopontin by allowing it to adopt a more native conformation permitted a greater degree of endothelial cell adhesion.

3] CELLULAR RESPONSES TO CHANGE IN SURFACE CHEMISTRY, OTHER PROPERTIES AND THE IMPORTANCE OF PROTEIN ADSORPTION:

Studying cell responses to surface chemistry aids the study of measures to alleviate problems of iatrogenic reactions, thrombosis mediated by the surface, complementary system activation, infection due to device implantation, inflammatory response, phagocyte initiated oxidation, stress cracking, tissue fibrosis, wear debris deposition by suitable modification methodologies (Thevenot, Hu and Tang, 2008, pp.270-280). The magnitude and composition of the host protein adhering to a biomaterial, and the degree of exposure of inflammatory epitopes need to be controlled by surface chemistry modification (Thevenot, Hu and Tang, 2008, pp.270-280). Protein adsorption onto biomaterial determines the host immune and coagulation response to implants (Anderson, Bonfield and Ziats, 1990, pp.375-382).The surface wettability, hydrophobicity, and surface charge affect the protein adsorption. The events that occur after the implantation of a medical implant in the body include deposition of plasma proteins i.e., albumin, fibrinogen, IgG, fibronectin and the von Willebrand factor (Thevenot, Hu and Tang, 2008, pp.270-280). Thevenot and colleagues proved that mounting heparin and PEO to implants increased blood compatibility. Protein grafting to alter the surface chemistry caused the loss of motility of protein on the surface and may also cause toxic monomer residue (Thevenot, Hu and Tang, 2008, pp.270-280).

SAMs are capable of controlling the density and conformation of the single/multiple functional groups. Well-defined surfaces with ordered functionalities of appropriate thermodynamic equilibrium are desired. In vivo studies conducted by Barbosa et al. deal with modification of surface chemistry using SAMs (Barbosa et al., 2006, pp.737-743). The generally used SAMs include the Au and Ag based thiols (Barbosa et al., 2006, pp.737-743).

Plasma modification using RF, microwaves, electrons, and arc discharge modifies the surfaces without considering the geometry (Thevenot, Hu and Tang, 2008, pp.270-280). This affects the protein adsorption, denaturation, and epitope exposure. Surface chemistry- induced complement cascades of classical and alternative pathways have been explained (Craddock et al., 1977, pp.879-888). Complement cascades are capable of recruitment and activation of phagocytes in the host tissue. This leads to eventual adherence /activation of leucocytes in the host (Craddock et al., 1977, pp.879-888). IgG underwent denaturation on hydrophobic surfaces due to greater exposure of active sites (Thevenot, Hu and Tang, 2008, pp.270-280). The –OH functionalised surfaces were found to have greater leucocyte response. Hydrophilic surfaces possessing low interfacial energy decreased the protein adsorption, cell attachment and blood compatibility (Chen et al., 2005, pp. 307-311). FN adsorption decreased, albumin adsorption increased and platelet adhesion decreased in hydrophilic surfaces (Lee et al., 1998, pp.180-186). The leucocyte adhesion inhibition, significant decrease in macrophage fusion, cytokine secretion and inflammatory reaction are initiated by –OH functional groups. The –CH3 functionalisation of a surface increased leucocyte adhesion. The –COOH functionalised surfaces increased focal adhesion, reduced osteoblast differentiation, matrix mineralisation, and a reduction in myoblast differentiation (Ertel, Ratner and Horbett, 1990, pp.1637-1659). Contradictory results and findings exist in the case of –OH functionalised surfaces (Liu et al., 2005, pp.23-31) that indicate its reduced plasma protein adsorption and higher platelet compatibility via the protein repelling nature due to neutrality of the charge.

4] IMPLANT SELECTION BASED ON SURFACE CHEMISTRY:

Ideal implants are characterised by a dynamic surface chemistry whereby the induction of histological changes which are initiated at the implant surface are those that normally occur if the implant was absent (Clark, Hench and Paschall, 1976, pp.161-174). The validity of this theory was tested and proved by Clark and co-workers who conducted in vivo tests using bioglass implants on rat and sheep models. The bone implant studies conducted prove that excess/deficient surface ion concentration produced negative osteogenesis and fixation results (Clark, Hench and Paschall, 1976, pp.161-174). The studies conducted by Clark and colleagues hinted at various aspects of cell responses to changes in surface chemistry of hard as well as soft tissues. The ideas postulated are that the ions released from the implant surfaces of bioglass /bioglass ceramic are required for osteogenesis (Clark, Hench and Paschall, 1976, pp.161-174). Time-dependant pH alterations at the biomaterial surfaces of bioglass are necessary for osteogenesis. The results were postulated using a young rabbit tibia with enhanced metabolism and an old rat femur of impaired and decreased metabolism. Structural alterations of ultra-level magnitude at the orthopaedic implant surface facilitate the bonding of collage fibril and mucopolysaccharide with inorganic gel, resulting in matrix and bone mineralisation (Clark, Hench and Paschall, 1976, pp.161-174).

5] APPLICATIONS IN THE MEDICAL DEVICE INDUSTRY:
This section deals briefly with the applications of surface chemistry responses to altering the cell responses practised in the medical device industry. Orthopaedic implants of various types have been coated with antibacterial HAP coating functionalised surfaces to promote tissue integration without favouring bacterial colonisation. These coatings are HAP coatings functionalised onto an orthopaedic implant usually of Ti/stainless steel with Ag ions irreversibly implanted into it. The surface chemistry modification was developed on a commercial scale by Medicoat (www.medicoat.ch/EN/home.html). The utilisation of bioresorbable polymeric coatings for lubrication of interventional catheters or drug elution coatings for permanent implants has been developed by Bayer Material Science Division and is being commercialised (www.bayer.com/en/bayer-‌materialscience.aspx). The studies conducted at the Brown University in collaboration with the Coulter and Hermann foundation proved that drugs immobilised in polydexamethasone films electrodeposited on AuPd-Ti implants and MWNT-Ti implants enhanced osteoblast adhesion in vitro and inhabited fibroblast adhesion compared to plain Ti implants.
Pareta et al. has proved that NCD-coated titanium can be a favourable material to prolong the wear properties of current titanium implants at articulating and non-articulating interfaces. This can also promote bone-implant bonding, enhancing the implant longevity.

Surmodics, a regenerative technology-based company has commercialised the development of surface chemistry modifying coating to ensure the better performance of medical devices. The hydrophilic passivating Rejoice coating was developed to reduce the adsorption of fibrinogen from blood (Hupfer, no date). The Applause heparin involved in active inhibition of thrombotic activity was used for thrombosis reduction of PE implants. The reliability of the technique was tested on a porcine carotid crush model using Applause heparin and Bravo products and proved that a dry thrombus mass was significantly reduced on coated PE implant devices (Hupfer, no date). The Finale haemostatic coating reduced endovascular leaks in AAA endovascular stents and grafts. A fibrin platelet framework effectively sealed polyester graft fibric pores encapsulated the leak five minutes post-placement (Hupfer, no date). This can be used in vascular grafts, stent grafts, cardiac patches, metallic devices such as aneurism clips, vascular stents (Hupfer, no date).

6 SUMMARY:
Cell responses to surface chemistry determine the performance of a biomaterial implant in the body. The literature associated with this phenomenon is discussed in Sections 1-5. Surface chemistry modifications can enhance or inhibit the cell adhesions, focal adhesion activity, integrin binding, cell proliferation and differentiation, matrix mineralisation, platelet adhesion etc. This can improve the surface properties of a biomaterial. Protein adsorption forms the major cell response to biomaterials which has different mechanisms.