For osseointegration to work, bone cells must make contact with the implant surface. Osteoblasts are those cells that build new or repair damaged bone. This is a complex process called osteogenesis and starts with the adhesion of osteoblasts to existing bone or the implant surface. Further events involve bone cell proliferation and differentiation, expression of bone cell-specific enzymes and products, secretion, and deposition of the organic bone matrix. When a bone cell, floating in interstitial fluid or blood, approaches the implant it only “feels” the device surface, not the bulk material. This surface displays charged and hydrogen-bond groups and lipophilic patches originating from either implant or adsorbed extracellular matrix. This matrix originates from adsorption of proteins from surrounding tissue fluid or blood and is also secreted by the bone cells. Typical matrix ligands are collagen type I and fibronectin. Some implants display those bio-adhesive ligands via their surface coating (Cohen et al., 2004; Garcia and Reyes, 2005). Initially, there are weak and temporary interactions between charged or polar molecules of cell membrane and implant surface. Over time, those interactions can become stronger and more specific leading to permanent cell adhesion via binding of adhesion receptors (e.g., integrins, cadherins) located on the osteoblast cell membrane to extracellular matrix ligands present on the implant surface. After binding to an extracellular matrix ligand, the integrins associate with the actin cytoskeleton and cytoplasmic plaque proteins (e.g., vinculin) to form focal adhesions (Gumbiner, 1996). At a focal adhesion, the distance between cell membrane receptor and ligand on the implant surface drops to only 15 nm (Cohen et al., 2004). Focal adhesions are crucial for the spreading and flattening of a cell that is important to gain strong adhesion. Together with growth factor receptors, focal adhesions also trigger integrin-activated signaling cascades leading to osteoblast differentiation and physiological function (Garcia and Reyes, 2005). The establishment of integrin receptor–matrix ligand occurs within minutes after the initial surface recognition. Adhesion with proper cell spreading then takes several hours during which the cell–implant contact area increases 100-fold (Cohen et al., 2004). The adhesion process can be greatly influenced by the specific physico-chemical properties – such as charges, chemistry, wettability, roughness – and nano-structuring of the implant surface (Monsees et al., 2005; Ozkucur et al., 2009b; Novaez et al., 2010). Thus, it is extremely important to compare those parameters with the outcome of the biocompatibility tests.

Implanted devices are prone to biofilm formation. A biofilm is a thin but robust layer of microorganisms, such as bacteria, fungi, and yeast that adhere to the implant surface similar to the dental plaque sticking to the teeth (Actis et al., 2013; Kargupta et al., 2014). These microorganisms can cause serious infections around the implant initiating progressive bone loss leading to loosening or even loss of the implant (Wood et al., 2004). In case of titanium implants, the surface oxide coating achieves an amalgamation with the superficial gum that hinders the invasion of oral microorganisms. However, the amount of bacterial colonization can be influenced by implant surface coating or structuring (Table 1). For example, a rougher surface supports bacterial growth and provides protection from saliva and cleansing by the tongue, whereas a smoother surface limits the adhesion of bacteria (Rimondini et al., 1997). Because of this interaction, antibacterial activity tests should also be considered when testing the biocompatibility of a novel coating. Modern antimicrobial coatings are designed to prevent biofilm formation by, e.g., preventing bacterial adhesion, killing bacteria, or by accelerating osteoblast adhesion (reviewed in Hasan et al., 2013; Kargupta et al., 2014).

There are two principal classes of cells: established cell lines and primary cells. Established cell lines are considered the standard for similar research that makes comparison of results and standardization of procedures possible. Cell lines can be bought commercially from trusted sources, are easy to work with, and can be used for many years. However, because these cells can divide continuously, they may not reflect the true physiology of the original cell. By contrast, primary cells must be isolated directly from the body and can be kept in culture for only a limited time. This makes them difficult and expensive to work with; however, the results are much closer to the in vivo situation. Often used cell types are immortalized neonatal mouse osteoblasts (MC3T3E1), human osteosarcoma (SaOS-2 or MG-63), primary rat calvaria cells, or L929 mouse fibroblasts (Meyer et al., 2005; Monsees et al., 2005; Bierbaum et al., 2006; Subramanian et al., 2015).

Cytotoxicity may be tested using an extract or the implant material itself. The conditions of extraction should meet the intended clinical use. Suitable eluents are cell culture media or physiological saline. The tests must include a negative control (culture medium) and positive control (e.g., 6% DMSO in culture medium). Cells must be incubated for a minimum period of 24 h. For testing cytotoxicity in direct cell contact, cells are applied to the surface of suitable samples under sterile conditions and then incubated for 1–3 days. Alternatively, the implant sample is carefully placed on top of the cell layer. Other approved tests are the Agar and the Filter Diffusion assay. Both are indirect contact tests that evaluate if substances from the implant can be dissolved and move through a thin barrier (agar layer or inert filter) toward the cells.

For transparent samples, cell numbers and morphology can be checked via simple light microscopy. Living cells should have their typical form and are well attached and spread; they usually form clusters (Subramanian et al., 2015). Damaged or dying cells develop vacuoles, may shrink, become rounder in appearance, and eventually detach from the surface. Thus, on a sub-optimal surface, cell numbers are lower. For non-transparent samples, cells can be visualized using an epifluorescence or scanning electron microscope (Ozkucur et al., 2009a). For quantitative measurement of cytotoxicity, suitable parameters are inhibition of cell proliferation, numbers of dead and live cells, reduction of vital dyes, and release of cytoplasmic enzymes as a result of cell membrane damage. The MTT assay measures the activity of mitochondrial enzymes, which not only reflects the number of viable cells present (used as proliferation assay) but also mirrors the degree of mitochondrial activity that indicates cellular stress or dying cells (used as cytotoxicity test). Technically, this assay works by reduction of the yellow tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a purple insoluble formazan. This color increase can be measured using a photometer. Ozkucur et al. (2009a) showed significantly higher MTT-values, thus more living cells attached on metal-coated polyurethane compared to the uncoated surfaces. Also, the titanium thin film seems to be more favorable for adhesion than the zirconium coating. Other related tetrazolium dyes are XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), and WSTs (water-soluble tetrazolium salts). These colorimetric assays provide average values off many thousands cells but do not give any information about the fate of individual cells. Here, Live/Dead assays using a pair of fluorophores are the better choice. The general principle is that one of these fluorophores can enter the living cell (intact membrane), whereas the other one cannot. The second dye can only enter if the cell membrane is compromised indicating a dead or dying cell. This second dye usually then binds to DNA in the cell nuclei. An example is the calcein-AM – ethidium homodimer, EthD-1 assay that shows living cells in green, whereas dead cells emit red fluorescence (Huang et al., 2016).

The sequence leading to cell adhesion and spreading gives us some important parameter for additional characterization. There are several cellular components that can be visualized, e.g., using specific fluorescent dye-tagged antibodies. Often used targets are the actin stress fibers that mark the cell morphology; vinculin, a protein located in focal adhesions; and DNA to visualize the cell nuclei. In this way, different degrees of cell adhesion and spreading can be clearly distinguished (Monsees et al., 2005). Ozkucur et al. (2009a) depicted human umbilical vein endothelial cells grown on titanium thin-film-coated polyurethane: cells are well spread with regularly organized actin cytoskeleton and many focal adhesion contacts. By contrast, cell numbers and spreading on the uncoated polyurethane were very poor. Similarly, adhesion and spreading of endothelial cells is poor on polyurethane, but is dramatically improved on a 30-nm titanium film as shown with scanning electron microscopy. The more sophisticated Wound-Healing assay assesses cell proliferation, actin cytoskeleton reorganization, dissolving/renewing of focal adhesion points, and migration. Here, cells are grown on the implant surface then an open gap is created by removing cells via scratching through the confluent cell monolayer. The time for the cells to close this “wound” by proliferation, spreading, and moving is monitored (Oprea et al., 2003; Bindschadler and McGrath, 2007).

As the implant surface properties can also influence osteoblast differentiation and, thus, ultimately their physiological function, more specialized investigations should be considered. Typical osteoblast-specific parameters include activity of alkaline phosphatase enzyme, secretion of collagen type I, osteopontin or osteocalcin, or formation of calcium phosphate deposits (Hempel et al., 2004; Meyer et al., 2005). Alkaline phosphatase activity can be determined, e.g., via histochemical staining and fluorescence microscopy. Monsees et al. (2005) noted regular alkaline phosphatase activity in SaOS-2 osteoblasts attached to smooth or structured titanium thin films. By contrast, surface composition greatly influenced osteopontin expression.

Material degradation or corrosion within the body can lead to loosening and failure of the implant. The impact of body fluids on the degradation of metallic implants can be modeled using artificial saliva, blood serum, and salt solution (media) normally used for tissue culture at 37°C for longer periods of time (days or weeks) (ISO 10993 Part 15). Parameters investigated may include changes in weight or hardness of the implant material, electrochemical behavior, changes of the surface, by means of profilometry, scanning electron or atomic force microscopy, and nature and amount of metal ions leaching from the implant assessed by X-ray diffraction and atomic emission spectroscopy (Wetzel et al., 2008; Lesniewicz et al., 2010). It is worthwhile to also compare potential biodegradation with implants that have been in contact with cells for the same time and conditions. Depending on the surface coating and the underlying bulk material (metal alloy, ceramics, or polyurethane), the presence of cells may protect the surface, e.g., by secreting extracellular matrix proteins (Ozkucur et al., 2009a) or may accelerate especially polymer degradation by releasing hydrolytic enzymes (Ozkucur et al., 2009b).

Microorganisms often used in testing the anti-microbiological activity of implants include bacteria that can be present in the oral cavity, e.g., Staphylococcus aureus and Pseudomonas aeruginosa (Gram-negative), and Streptococcus sanguinis and Escherichia coli (Gram-positive) (Pye et al., 2009). Two often used tests are the Agar Disc Diffusion assay and the Direct Contact method. In the agar diffusion assay, the respective single strain of a microorganism is spread on solidified agar medium. Then the implant sample is gently pressed on the agar and incubated for 24 h at 37°C. Any antibacterial activity is measured as a zone of inhibition around the sample disk. A standard implant surface serves as “negative control,” whereas an appropriate antibiotic serves as positive control (Subramanian et al., 2015). For direct contact method, media containing the respective bacteria are placed onto round implant samples and then incubated at 37°C for 24 h. Alternatively, implant samples can be incubated on a shaker with the respective bacteria suspension. Adhered bacteria populations can be visualized after appropriate staining using a stereo-optical or epifluorescence microscope or processed for scanning electron microscopy (Chung et al., 2006; Patenge et al., 2012).