Microbial infection and insufficient tissue formation are considered to be the two main causes of dental implant failure. Novel studies have focused on designing dual-functional strategies to promote antibacterial properties and improve tissue cell response simultaneously. In this study, we investigated the antibacterial properties and cytocompatibility of silver nitrate (AgNO₃) and strontium acetate (SrAc) in a mono-culture setup for dental application. Additionally, we defined the therapeutic window between the minimum inhibitory concentration against pathogenic bacteria and maximum cytocompatible dose in the case of combined applications in a co-culture setup. Antibacterial properties were screened using Aggregatibacter actinomycetemcomitans and cell response experiments were performed with osteoblastic cells (MC3T3) and fibroblastic cells (NIH3T3). The osteoinductive behavior was investigated separately on MC3T3 cells using alizarin red staining. A therapeutic window for AgNO₃ as well as SrAc applications could be defined in the case of MC3T3 cells while the cytocompatibility of NIH3T3 cells was compromised for all concentrations with an antibacterial effect. However, the combined application of AgNO₃/SrAc caused an enhanced antibacterial effect and opened a therapeutic window for both cell lines. Enhanced mineralization rates could be observed in cultures containing SrAc. In conclusion, we were able to demonstrate that adding SrAc to AgNO₃ not only intensifies antibacterial properties but also exhibits bone inductive characteristics, thereby offering a promising strategy to combat peri-implantitis and at the same time improve osseointegration in implant therapy.

Magnesium alloys are presently under investigation as promising biodegradable implant materials with osteoconductive properties. To study the molecular mechanisms involved, the potential contribution of soluble magnesium corrosion products to the stimulation of osteoblastic cell differentiation was examined. However, no evidence for the stimulation of osteoblast differentiation could be obtained when cultured mesenchymal precursor cells were differentiated in the presence of metallic magnesium or in cell culture medium containing elevated magnesium ion levels. Similarly, in soft tissue no bone induction by metallic magnesium or by the corrosion product magnesium hydroxide could be observed in a mouse model. Motivated by the comparatively rapid accumulation solid corrosion products physicochemical processes were examined as an alternative mechanism to explain the stimulation of bone growth by magnesium‐based implants. During exposure to physiological solutions a structured corrosion coat formed on magnesium whereby the elements calcium and phosphate were enriched in the outermost layer which could play a role in the established biocompatible behavior of magnesium implants. When magnesium pins were inserted into avital bones, corrosion lead to increases in the pull out force, suggesting that the expanding corrosion layer was interlocking with the surrounding bone. Since mechanical stress is a well‐established inducer of bone growth, volume increases caused by the rapid accumulation of corrosion products and the resulting force development could be a key mechanism and provide an explanation for the observed stimulatory effects of magnesium‐based implants in hard tissue. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 697–709, 2017.

Biomaterial-associated Pseudomonas aeruginosa biofilm infections constitute a cascade of host immune reactions ultimately leading to implant failure. Due to the lack of relevant in vivo biofilm models, the majority of the studies report host immune responses to free-living or planktonic bacteria, while bacteria in clinical situations live more frequently as biofilm communities than as single cells. The present study investigated host immune responses to biomaterial-associated P. aeruginosa biofilms in a clinically relevant mouse model. Previously, we reported metallic magnesium, a prospective biodegradable implant, to be permissive for bacterial biofilm in vivo even though it exhibits antibacterial properties in vitro . Therefore, magnesium was employed as biomaterial to investigate in vivo biofilm formation and associated host immune responses by using two P. aeruginosa strains and two mouse strains. P. aeruginosa formed biofilm on subcutaneously implanted magnesium disks. Non-invasive in vivo imaging indicated transient inflammatory responses at control sites, whereas robust prolonged interferon- β (IFN- β ) expression was observed from biofilm in a transgenic animal reporter. Furthermore, immunohistology and electron microscopic results showed that bacterial biofilms were located in 2D immediately on the implant surface and at a short distance in the adjacent tissue. These biofilms were surrounded by inflammatory cells (mainly polymorphonuclear cells) compared to the controls. Interestingly, even though the number of live bacteria in various organs remained below detectable levels, splenomegaly indicated systemic inflammatory processes. Overall, these findings confirmed the resistance of biofilm infections in vivo to potentially antibacterial properties of magnesium degradation products. In vivo imaging and histology indicated the induction of both local and systemic host inflammatory responses to P. aeruginosa biofilms. Even though the innate host immune defenses could not eliminate the local infection for up to two weeks, there was no apparent systemic bacteremia and all the animals investigated survived the infection.

Cytokine profiles are often perturbed after infections of medical implants. With a non-invasive in vivo imaging system, we report in a mouse model that interferon expression after infection of subcutaneous implants with Streptococcus oralis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Treponema denticola (alone or as a combination) was species-specific, persisted longer in the presence of implants, and notably decreased upon dual species infections. This type I interferon expression disappeared within two weeks; however, histology of implant–tissue interface indicated high recruitment of immune cells even after three weeks. This was suggestive that biomaterial-associated infections could have prolonged effects, including the systemic stimulation of inflammatory cytokines. The present study investigated the systemic impact of this chronic peri-implant inflammation on the systemic expression of inflammatory cytokines (23) using a multiplex assay. Initially, the cytokine measurement in murine fibroblasts exposed to periodontal pathogens remained limited to the expression of five cytokines, namely, IL-6, G-CSF, CXCL-1/KC, MCP-1 (MCAF), and IL-12 (p40). The systemic determination of cytokines in mice increased to 19 cytokines (IL-1α, IL-2, IL-3, IL-5, IL-6, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-17A, CCL-11/Eotaxin, G-CSF, IFN-γ, CXCL1/KC, MCP-1 (MCAF), MIP-1α/CCL3, MIP-1β/CCL4, CCL5/RANTES, and TNF-α). Systemic induction of cytokines was species-specific in the mouse model. The cytokine induction from infected implants differed significantly from sole tissue infections and sterile implants. Notably, systemic cytokine induction decreased after infections with dual species compared to single species infections. These findings describe the systemic effect of chronic peri-implant inflammation on the systemic induction of inflammatory cytokines, and this effect was strongly correlated to the type and composition of initial infection. Systemic modulations in cytokine expression upon dual species infections exhibit an exciting pattern that might explain the complications associated with biomaterial-related infection in patients. Moreover, these findings validate the requirement of multispecies infections for pre-clinical studies involving animal models.

Type I interferon-beta (IFN-β) is a crucial component of innate and adaptive immune systems inside the host. The formation of bacterial biofilms on medical implants can lead to inflammatory diseases and implant failure. Biofilms elicit IFN-β production inside the host that, in turn, restrict bacterial growth. Biofilms pose strong antibiotic resistance, whereas surface modification of medical implants with antibacterial agents may demonstrate strong antimicrobial effects. Most of the previous investigations were focused on determining the antibacterial activities of implant surfaces modified with antibacterial agents. The present study, for the first time, measured antibacterial activities and IFN-β expression of titanium surfaces along with silver or tetracycline inside co-culture and mouse models. A periodontal pathogen: Aggregatibacter actinomycetemcomitans reported to induce strong inflammation, was used for infection. Silver and tetracycline were added to the titanium surface using the heat evaporation method. Macrophages showed reduced compatibility on titanium surfaces with silver, and IFN-β expression inside cultured cells significantly decreased. Macrophages showed compatibility on implant surfaces with tetracycline, but IFN-β production significantly decreased inside seeded cells. The decrease in IFN-β production inside macrophages cultured on implant surfaces with silver and tetracycline was not related to the downregulation of Ifn-β gene. Bacterial infection significantly upregulated mRNA expression levels of Isg15, Mx1, Mx2, Irf-3, Irf-7, Tlr-2, Tnf-α, Cxcl-1, and Il-6 genes. Notably, mRNA expression levels of Mx1, Irf7, Tlr2, Tnf-α, Cxcl1, and Il-6 genes inside macrophages significantly downregulated on implant surfaces with silver or tetracycline. Titanium with tetracycline showed higher antibacterial activities than silver. The in vivo evaluation of IFN-β expression around implants was measured inside transgenic mice constitutive for IFN-β expression. Of note, the non-invasive in vivo imaging revealed a significant decrease in IFN-β expression around subcutaneous implants with silver compared to titanium and titanium with tetracycline in sterile or infected situations. The histology of peri-implant tissue interfaces around infected implants with silver showed a thick interface with a significantly higher accumulation of inflammatory cells. Titanium implants with silver and tetracycline remained antibacterial in mice. Findings from this study unequivocally indicate that implant surfaces with silver decrease IFN-β expression, a crucial component of host immunity.