In Pursuit of Alternatives to Poly(ethylene glycol) as Protein-Repellent but Cell-Adhesive Surface Coatings for Biomaterials

Biomaterials are indispensable in health care. They are used in a plethora of applications, whenever the function of an organ or tissue needs to be supported, restored or replaced temporarily or permanently. The fate of any device interacting with the human body strongly depends on the amount and composition of the initially adsorbed protein layer. It governs, for instance, bacterial adhesion, potentially leading to a bacterial infection, and tissue cell attachment, allowing for successful tissue integration of an implant. As protein adsorption is the first step in a cascade of interactions between biomaterials and living organisms, it is crucial to further understand how it is influenced by the biomaterial surface. Thus, this PhD thesis focuses on the investigation of surface properties and their effect on biological systems, pursuing two goals: Firstly, the research was aimed at developing a deeper understanding how protein adsorption is related to macroscopically determined surface characteristics. In addition to that, it was sought to develop novel surface modifications, which ideally combine protein-repellent with cell-adhesive properties for later application in dental or orthopedic implants. To this purpose, a large variety of surface modifications has been developed and synthesized on appropriate model substrates using silane chemistry. The research of this thesis will be divided into four parts, all sharing a similar structure. At first, the successful synthesis of the respective surface coatings was confirmed via X-ray photoelectron spectroscopy (XPS), infrared (IR) spectroscopy and water contact angle measurements. In addition to that, fundamental physicochemical properties of the surface modifications were characterized: The surface free energy (SFE) and dynamic wetting behavior were analyzed via static contact angle measurements or tensiometry respectively. The zeta potential of the surface functionalizations was derived from electrophoresis or streaming current measurements. Furthermore, the interaction of those surface modifications with biological systems was investigated, including the quantitative or qualitative analysis of protein adsorption or the study of initial cell adhesion. In the first chapter, protein adsorption was investigated on a variety of self-assembled monolayers (SAMs) with different terminal functional groups. Covering a broad range of surface wettabilities, SFEs and zeta potentials, the surfaces’ properties were shown to substantially influence qualitative and quantitative protein adsorption from the biofluids human saliva and human serum. Whereas some single proteins, most prominently lysozyme, clearly followed basic physicochemical rules in their adsorption behavior, no such dependence could be observed for the majority of proteins. The amounts and compositions of the adsorbed protein layers were clearly shaped by the characteristics of the surface, but no simple relations between surface properties and protein adsorption could be revealed. The second chapter focuses on a group of surface modifications, which are related to the chemistry of the poly(amido amine) (PAMAM) dendrimer. Those six surface coatings all possess terminal and inner amine groups, but they differ with respect to the presence or absence of inner amide groups and with respect to their structure, being either short-chained oligomers, linear polymers or dendrimers. These functionalizations all exhibited a moderately hydrophilic behavior, but they differed significantly in their electrokinetic behavior. Protein adsorption from single protein solutions as well as from human saliva and fetal bovine serum (FBS) was found to be strongly governed by the zeta potential. Both linear polymers, characterized by their flexibility and hydrophilicity, exhibited protein-repellent behavior. In addition to the PAMAM-derived coatings, further surface modifications with osmolyte motifs were developed, inspired by their unique protein-stabilizing function in nature. Immobilization of sulfobetaine or amine oxide groups led to hydrophilic surface coatings with negative zeta potentials under physiological conditions. The amine oxide modification, based on the osmolyte trimethylamine N-oxide, exhibited excellent protein-repellency. In the last chapter, three amine-based modifications, representing the three different structural motifs, were immobilized on titanium substrates for cell experiments, analyzing the behavior, i.e. spreading, morphology, actin cytoskeleton and cell cycle, of osteoblastic MG-63 cells up to 24 h. In general, large differences in cell behavior were observed after 1 h of cultivation, which almost vanished after 24 h of cultivation. A drastically increased initial cell spreading was observed on the dendrimer coating, which was characterized by a large density of surface amine groups and a strongly positive zeta potential under physiological conditions

Enhancement of Intracellular Calcium Ion Mobilization by Moderately but Not Highly Positive Material Surface Charges

Electrostatic forces at the cell interface affect the nature of cell adhesion and function; but there is still limited knowledge about the impact of positive or negative surface charges on cell-material interactions in regenerative medicine. Titanium surfaces with a variety of zeta potentials between −90 mV and +50 mV were generated by functionalizing them with amino polymers, extracellular matrix proteins/peptide motifs and polyelectrolyte multilayers. A significant enhancement of intracellular calcium mobilization was achieved on surfaces with a moderately positive (+1 to +10 mV) compared with a negative zeta potential (−90 to −3 mV). Dramatic losses of cell activity (membrane integrity, viability, proliferation, calcium mobilization) were observed on surfaces with a highly positive zeta potential (+50 mV). This systematic study indicates that cells do not prefer positive charges in general, merely moderately positive ones. The cell behavior of MG-63s could be correlated with the materials’ zeta potential; but not with water contact angle or surface free energy. Our findings present new insights and provide an essential knowledge for future applications in dental and orthopedic surgery. © Copyright © 2020 Gruening, Neuber, Nestler, Lehnfeld, Dubs, Fricke, Schnabelrauch, Helm, Müller, Staehlke and Nebe

Comparison of Protein-Repellent Behavior of Linear versus Dendrimer-Structured Surface-Immobilized Polymers

For many biomedical applications, material surfaces should not only prevent unspecific protein adsorption and bacterial attachment as in many other applications in the food, health, or marine industry, but they should also promote the adhesion of tissue cells. In order to take a first step toward the challenging development of protein and bacteria-repelling and cell-adhesion-promoting materials, polyamine and poly(amido amine) surface coatings with terminal amine groups and varying structure (dendrimer, oligomer, polymer) were immobilized on model surfaces via silane chemistry. Physicochemical analysis showed that all modifications are hydrophilic (contact angles <60 degrees) and possess similar surface free energies (SFEs, similar to 46-54 mN/m), whereas their amine group densities and zeta potentials at physiological conditions (pH 7.4) varied greatly (-50 to +75 mV). In protein adsorption experiments with single proteins (human serum albumin (HSA) and lysozyme) as well as complex physiological fluids (fetal bovine serum (FBS) and human saliva), the amounts of adsorbed protein were found to correlate strongly with the zeta potential of the surface coatings. Both modifications based on linear polymers exhibited good protein repellency toward all proteins examined and are thus promising for testing in cell adhesion studies

Terminal chemical functions of polyamidoamine dendrimer surfaces and its impact on bone cell growth

Besides their use for drug and gene delivery, dendrimer molecules are also favorable for the design of new surface coatings for orthopedic and dental implants due to the wide variety of functional terminal groups and their multivalent character. The purpose of this work was to observe how covalently immobilized polyamidoamine (PAMAM) dendrimer molecules with different terminal chemical groups influenced serum protein adsorption and osteoblast behavior. To this end, fifth-generation PAMAM dendrimers were immobilized on silicon surfaces with an anhydride-containing silane coupling agent which results in positively charged terminal NH2-groups. Coatings with a net negative charge were generated by introduction of terminal CO2H- or CH3- groups. Surface characterization was performed by static and dynamic contact angle and zeta potential. The in vitro studies with human MG-63 osteoblastic cells focused on cell adhesion, morphology, cell cycle, apoptosis and actin formation within 24 h. This work demonstrated that cell growth was dependent on surface chemistry and correlated strongly with the surface free energy and charge of the material. The positively charged NH 2 surface induced tight cell attachment with well-organized actin stress fibers and a well spread morphology. In contrast, CO2H- and CH3-functional groups provoked a decrease in cell adhesion and spreading and indicated higher apoptotic potential, although both were hydrophilic. The knowledge about the cell-material dialogue is of relevance for the development of bioactive implants in regenerative medicine

Saliva and Serum Protein Adsorption on Chemically Modified Silica Surfaces

Biomaterials, once inserted in the oral cavity, become immediately covered by a layer of adsorbed proteins that consists mostly of salivary proteins but also of plasma proteins if the biomaterial is placed close to the gingival margin or if it becomes implanted into tissue and bone. It is often this protein layer, rather than the pristine biomaterial surface, that is subsequently encountered by colonizing bacteria or attaching tissue cells. Thus, to study this important initial protein adsorption from human saliva and serum and how it might be influenced through chemical modification of the biomaterial surface, we have measured the amount of protein adsorbed and analyzed the composition of the adsorbed protein layer using gel electrophoresis and western blotting. Here, we have developed an in vitro model system based on silica surfaces, chemically modified with 7 silane-based self-assembled monolayers that span a broad range of physicochemical properties, from hydrophilic to hydrophobic surfaces (water contact angles from 15° to 115°), low to high surface free energy (12 to 57 mN/m), and negative to positive surface charge (zeta potentials from –120 to +40 mV at physiologic pH). We found that the chemical surface functionalities exerted a substantial effect on the total amounts of proteins adsorbed; however, no linear correlation of the adsorbed amounts with the physicochemical surface parameters was observed. Only the adsorption behavior of a few singular protein components, from which physicochemical data are available, seems to follow physicochemical expectations. Examples are albumin in serum and lysozyme in saliva; in both, adsorption was favored on countercharged surfaces. We conclude from these findings that in complex biofluids such as saliva and serum, adsorption behavior is dominated by the overall protein-binding capacity of the surface rather than by specific physicochemical interactions of single protein entities with the surface

Enhancement of Intracellular Calcium Ion Mobilization by Moderately but Not Highly Positive Material Surface Charges

Electrostatic forces at the cell interface affect the nature of cell adhesion and function; but there is still limited knowledge about the impact of positive or negative surface charges on cell-material interactions in regenerative medicine. Titanium surfaces with a variety of zeta potentials between -90 mV and +50 mV were generated by functionalizing them with amino polymers, extracellular matrix proteins/peptide motifs and polyelectrolyte multilayers. A significant enhancement of intracellular calcium mobilization was achieved on surfaces with a moderately positive (+1 to +10 mV) compared with a negative zeta potential (-90 to -3 mV). Dramatic losses of cell activity (membrane integrity, viability, proliferation, calcium mobilization) were observed on surfaces with a highly positive zeta potential (+50 mV). This systematic study indicates that cells do not prefer positive charges in general, merely moderately positive ones. The cell behavior of MG-63s could be correlated with the materials' zeta potential; but not with water contact angle or surface free energy. Our findings present new insights and provide an essential knowledge for future applications in dental and orthopedic surgery