(IFN-
induction. The results showed that inflammatory signals can be detected by in vivo imaging after subcutaneous implantation of biocompatible or immune stimulatory implants. However, there were specific differences depending upon the assay system. The response to inflammatory proteases and cell growth signaling molecules appeared delocalized and was difficult to assign to one of several implants in individual animals. On the other hand, the interferon response was locally focused and was highly specific for pathogens whereas no signal was detected in response to wounding or to biocompatible implant materials. In conclusion, of the various detection systems investigated, the transgenic interferon mouse model could be applied to monitor bacterial implant infections and will be useful to evaluate the efficacy of antimicrobial implant coatings.
Keywords
Implant materialInfectionInflammationIn vivo imagingMouse modelWound healingFluorescenceBioluminescence1 Introduction
Cell culture test conditions are comparatively quick, well defined and reproducible. Cell morphology and number can be used to evaluate cell compatibility of the material surface [1–3]. However, in vitro results cannot reliably be extrapolated to the in vivo situation. An important difference is for example, the presence of the immune system in vivo. Therefore, it is essential to test novel implant materials in vivo before clinical applications can be envisioned. This includes the evaluation of the tissue compatibility of the materials per se, the processing and surface structures and more recently, potential coatings that may be used for drug release purposes to improve the implant performance [4]. Mainly, such tests characterize the effects on the tissue surrounding the implant and the potential to induce immune reactions. For the detailed analysis of the tissue response to novel implant materials, histology can be used. Simple staining procedures can be used to visualize tissue damage, identify immune cells and the extent of granulation tissue is an indication for the degree of inflammation. Subsequently, the progress of the wound-healing reaction and the formation of fibrous capsule around implants can be evaluated. Staining with antibodies can be used for the highly specific detection of cells, including their differentiation and activation status. The major disadvantage of histological analysis is that solely a single predetermined time point can be analyzed for each implant and each animal. Alternatively, gene expression analysis has become a routine tool for obtaining detailed information about molecular events of the implant tissue interaction. This may serve to identify specific biological mechanisms and find ways to enhance this interaction or to improve the healing process. On the other hand, gene expression analysis generally gives only an average summary over processes in the tissue from many diverse cell types and complex interactions that take place between them. Gene expression analysis is an intensive method and similar to histological analysis, allows a single time point per implant or per animal [5].These methods preclude the monitoring of tissue responses to individual implants over time. Alternatively, in vivo imaging can be used advantageously to quantify and monitor tissue responses over an extended period of time in individual animals [6–12]. In this study, various clinically established implant materials were used for comparison. As biocompatible standard materials titanium and glass-ceramic implants were used. These are clinically used as dental, orthopedic or middle ear implants respectively [13–16]. In addition, inactivated bacteria were used as highly inflammatory and immune-stimulatory agents that could reflect implant infections.
As soon as an implant material comes into contact with body liquids such as blood or interstitial fluid, proteins are adsorbed on the implant surface [17]. It is however not clear to what extend this protein layer affects the subsequent interactions. In addition, inflammatory molecules are released by stressed or damaged cells and by reactions taking place during blood coagulation [18]. These molecules form a concentration gradient that recruits inflammatory cells from the blood circulation. In response, endothelial cells that line nearby blood vessels express adhesion molecules on the cell surface that allow the adhesion of circulating granulocytes and macrophages. These attach loosely, then adhere more firmly to the vessel walls and eventually penetrate them, guided by the concentration gradient they move through the tissue to the site of injury. The migration through the tissue requires the action of inflammatory proteases that degrade extracellular matrix components and lead to the loosening of cell–cell interactions. Polymorphonuclear neutrophils (PMNs) are the first cells that accumulate in the tissue within the first four hours after injury. These cells can recognize danger signals released from injured tissue via various pattern recognition receptors (PRRs). The signaling of these receptors can activate several inflammatory signaling pathways, there by promoting further inflammatory events and the recruitment of more cells from the blood circulation. Activated PMNs produce reactive oxygen species and proteases [19–22]. These reactions are a defense against foreign bodies which, depending on their nature may be degraded and superficial foreign bodies may be extruded. Any pathogens associated with the intrusion of a foreign body are attacked and, if successful, eradicated or at least locally contained until the adaptive immune system takes over. During excessive inflammation, these reactions, even lead to cell death and damage of the surrounding tissue. After a few hours monocytes are attracted from the blood circulation by the action of small peptides called chemokines that are released from PMNs and to a lesser degree by other cells. The monocytes mature to macrophages that control subsequent steps in the tissue, including the wound-healing reaction and the formation of a fibrotic capsule around the foreign body. Thereby, the inflammation is switched from the acute to the chronic stage (Fig. 1) [19, 23]. To establish a reliable testing system that can evaluate the inflammatory potential of novel implant materials we evaluated several strategies to visualize biomaterial-associated inflammatory reactions in vivo. Reactive oxygen species are produced by immune cells in response to inflammation. The oxidizing potential of these radicals was assessed. Hydrocyanines were used as chemical sensors which are oxidized to fluorescent cyanines in the presence of ROS. Cyanines can be visualized after excitation by their fluorescence in the near infra-red spectrum [24]. Alternatively, inflammatory proteases like cathepsins are produced by activated neutrophils or macrophages. The protease activity can be imaged using a specific probe that is hydrolyzed by cathepsins into a fluorescent product [25–27]. In a third approach, the activity of the extracellular lipase autotaxin was used to visualize an inflammatory process. Autotaxin activity generates a lipid signaling molecule that stimulates cell proliferation, cell migration and cell survival [29] and has been shown to be produced during lung inflammation [28]. For imaging, a fluorophore linked to a quencher by an autotaxin sensitive substrate was used. Upon cleavage with autotaxin, the fluorophore and its quencher are separated resulting in increased fluorescence efficiency. In addition, particularly in the early stages during the wound healing processes, implants are prone to colonization by bacteria. Bacterial infection induces cytokines like interferon-
(IFN-
. This has originally been discovered as part of the antiviral response but it is also induced by diverse bacteria [30, 31]. Bacterial infections could be visualized by using a transgenic mouse model in which the interferon(IFN)-
gene is replaced by a luciferase reporter gene that can be used for imaging purposes [32]. For imaging, heterozygous mice were used to allow IFN-
production from the wild type allele. The suitability of these in vivo imaging approaches was compared for the reliable evaluation and ranking of the biocompatibility of various implant materials and for detecting bacterial infections events in real time, respectively.
(IFN-. This has originally been discovered as part of the antiviral response but it is also induced by diverse bacteria [30, 31]. Bacterial infections could be visualized by using a transgenic mouse model in which the interferon(IFN)- gene is replaced by a luciferase reporter gene that can be used for imaging purposes [32]. For imaging, heterozygous mice were used to allow IFN- production from the wild type allele. The suitability of these in vivo imaging approaches was compared for the reliable evaluation and ranking of the biocompatibility of various implant materials and for detecting bacterial infections events in real time, respectively.Fig. 1
Implant-associated inflammation. Soon after implantation a protein layer is adsorbed on the implant surface (a) Recruitment of PMNs to the site of injury starts following injury and protein adsorption (b) Macrophages and monocytes are recruited at later stages (c)
2 Materials and Methods
2.1 Preparation of Hydrocyanine
To generate a chemical sensor for oxidative compounds for in vivo imaging 2 mg of indocyanine green (I2633, Sigma Aldrich) was dissolved in 4 ml of methanol (J.T. Baker
, Germany). 3 mg of sodium borohydride (Sigma Aldrich) was added to carry out reduction reaction. The solution was stirred continuously for 5 mins in absence of oxygen. Solvent was evaporated in the presence of vacuum using rotary evaporators. The dried powder was stored at 
C overnight. Before injection, the reduced hydrocyanine powder was dissolved in 2 ml of deionized water making up the final concentration to 1 mg/ml [24].
, Germany). 3 mg of sodium borohydride (Sigma Aldrich) was added to carry out reduction reaction. The solution was stirred continuously for 5 mins in absence of oxygen. Solvent was evaporated in the presence of vacuum using rotary evaporators. The dried powder was stored at C overnight. Before injection, the reduced hydrocyanine powder was dissolved in 2 ml of deionized water making up the final concentration to 1 mg/ml [24].2.2 Heat Inactivation of Staphylococcus Aureus
As a potent inflammation-inducing agent and as a substitute for infectious bacteria heat inactivated Staphylococcus aureus suspensions were prepared. The bacteria were streaked out on a Lysogeny broth medium (LB) agarose plate and incubated over night at 
. Single colonies were picked with a sterile needle and used to inoculate a liquid LB culture that was incubated on a rotary shaker at 180 rpm at 
. When the density of the culture reached an OD
of 0.1, 1 ml of the bacterial culture was centrifuged at maximum speed in an Eppendorf centrifuge for 5 mins at room temperature. The supernatant was discarded and the pellet was suspended in 1 ml of phosphate buffered saline (PBS), pH 7.0. For inactivation, the bacteria were first heated to 
for 15 mins and then stored on ice.
. Single colonies were picked with a sterile needle and used to inoculate a liquid LB culture that was incubated on a rotary shaker at 180 rpm at . When the density of the culture reached an OD of 0.1, 1 ml of the bacterial culture was centrifuged at maximum speed in an Eppendorf centrifuge for 5 mins at room temperature. The supernatant was discarded and the pellet was suspended in 1 ml of phosphate buffered saline (PBS), pH 7.0. For inactivation, the bacteria were first heated to for 15 mins and then stored on ice.2.3 Implant Preparation
For biocompatible and inflammatory implant preparation, respectively, plain material samples or samples coated with bacterial products were used. Porous implants were used to increase the carrier capacity and the stability of the coatings to prolong the release after implantation. Porous glass beads obtained from VitraPOR, Germany (Size 4 mm, Pore size-60 
m) were used as biocompatible implants. Inflammatory porous glass implants were prepared by soaking the beads for 2 min in heat inactivated Staphylococcus aureus suspensions and left to dry under ambient conditions. Porous titanium discs of 7 mm diameter and 2 mm thickness were prepared from micro-beads by an injection molding and sintering procedure. Magnesium discs with a diameter of 5 mm and height of 2 mm were prepared by extrusion of a rod followed by cutting off individual discs. Poly-L-lactic acid beads with a diameter of 5 mm were purchased from Good Fellow, England.
m) were used as biocompatible implants. Inflammatory porous glass implants were prepared by soaking the beads for 2 min in heat inactivated Staphylococcus aureus suspensions and left to dry under ambient conditions. Porous titanium discs of 7 mm diameter and 2 mm thickness were prepared from micro-beads by an injection molding and sintering procedure. Magnesium discs with a diameter of 5 mm and height of 2 mm were prepared by extrusion of a rod followed by cutting off individual discs. Poly-L-lactic acid beads with a diameter of 5 mm were purchased from Good Fellow, England.2.4 Subcutaneous Implantations in Mice
Wild-type BALB/c mice were obtained from Harlan–Winkelmann laboratories, Germany. Animals were housed under pathogen free conditions in a group of maximum five animals per cage. Mice were anesthetized by intraperitoneal injection of ketamine (10 mg/kg) and xylazine (4 mg/kg). The back was shaved using an electronic razor (Aesculap, Germany). An incision of 1 cm was made in the dorsal skin and a small pouch was made under the skin to insert the implant. The wound was closed by interrupted suturing using polyglactin filaments (Ethicon, Germany). Mock implantation was done by following the complete surgical procedure but without inserting any implant. All animal experiments were done in accordance with the regulations and with the approval from the local authorities Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), permission number 33.42502/ 07-10.5.
2.5 In Vivo Imaging of the Oxidation Potential
After anesthesia 30 
g of hydrocyanine solution was injected subcutaneously at the site of implantation. After 30 mins, fluorescent imaging was done in the near infrared spectrum using in vivo imaging system (IVIS200, Xenogen, USA). The excitation wavelength of hydrocyanines was 750 nm and the emission wavelength was 840 nm. Acquired images were corrected for the background using image math tool of living image software (version 4.3.1, Caliper life Sciences, 2012. In order to correct for the background, a region of interest was selected from a mouse without implants after addition of the fluorophore. The background value was calculated and automatically subtracted from the images by the software.
g of hydrocyanine solution was injected subcutaneously at the site of implantation. After 30 mins, fluorescent imaging was done in the near infrared spectrum using in vivo imaging system (IVIS200, Xenogen, USA). The excitation wavelength of hydrocyanines was 750 nm and the emission wavelength was 840 nm. Acquired images were corrected for the background using image math tool of living image software (version 4.3.1, Caliper life Sciences, 2012. In order to correct for the background, a region of interest was selected from a mouse without implants after addition of the fluorophore. The background value was calculated and automatically subtracted from the images by the software.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
