Sara Svensson

In vivo gene expression in response to anodically oxidized versus machined titanium implants

A quantitative polymerase chain reaction technique (qPCR) in combination with scanning electron microscopy was applied for the evaluation of early gene expression response and cellular reactions close to titanium implants. Anodically oxidized and machined titanium miniscrews were inserted in rat tibiae. After 1, 3, and 6 days the implants were unscrewed and the surrounding bone was retrieved using trephines. Both the implants and bone were analyzed with qPCR. A greater amount of cells, as indicated with higher expression of 18S, was detected on the oxidized surface after 1 and 6 days. Significantly higher osteocalcin (at day 6), alkaline phosphatase (at days 3 and 6), and cathepsin K (at day 3) expression was demonstrated for the oxidized surface. Higher expression of tumor necrosis factor-alpha (at day 1) and interleukin-1beta (at days 1 and 6) was detected on the machined surfaces. SEM revealed a higher amount of mesenchymal-like cells on the oxidized surface. The results show that the rapid recruitment of mesenchymal cells, the rapid triggering of gene expression crucial for bone remodeling and the transient nature of inflammation, constitute biological mechanisms for osseointegration, and high implant stability associated with anodically oxidized implants.

Osseointegration of titanium with an antimicrobial nanostructured noble metal coating.

UNLABELLED Nanometer scale surface features on implants and prostheses can potentially be used to enhance osseointegration and may also add further functionalities, such as infection resistance, to the implant. In this study, a nanostructured noble metal coating consisting of palladium, gold and silver, never previously used in bone applications, was applied to machined titanium screws to evaluate osseointegration after 6 and 12 weeks in rabbit tibiae and femurs. Infection resistance was confirmed by in vitro adhesion test. A qualitatively and quantitatively similar in vivo bone response was observed for the coated and uncoated control screws, using histology, histomorphometry and electron microscopy. The bone-implant interface analysis revealed an extensive bone formation and direct bone-implant contact. These results demonstrate that the nanostructured noble metal coating with antimicrobial properties promotes osseointegration and may therefore be used to add extra implant functionality in the form of increased resistance to infection without the use of antibiotics. FROM THE CLINICAL EDITOR The authors of this paper demonstrate that nanostructured noble metal coating of implants and prostheses used in orthopedic procedures promotes osseointegration and may be used to add extra implant functionality in the form of increased resistance to infection without the use of antibiotics.

Role of nanostructured gold surfaces on monocyte activation and Staphylococcus epidermidis biofilm formation

The role of material surface properties in the direct interaction with bacteria and the indirect route via host defense cells is not fully understood. Recently, it was suggested that nanostructured implant surfaces possess antimicrobial properties. In the current study, the adhesion and biofilm formation of Staphylococcus epidermidis and human monocyte adhesion and activation were studied separately and in coculture in different in vitro models using smooth gold and well-defined nanostructured gold surfaces. Two polystyrene surfaces were used as controls in the monocyte experiments. Fluorescent viability staining demonstrated a reduction in the viability of S. epidermidis close to the nanostructured gold surface, whereas the smooth gold correlated with more live biofilm. The results were supported by scanning electron microscopy observations, showing higher biofilm tower formations and more mature biofilms on smooth gold compared with nanostructured gold. Unstimulated monocytes on the different substrates demonstrated low activation, reduced gene expression of pro- and anti-inflammatory cytokines, and low cytokine secretion. In contrast, stimulation with opsonized zymosan or opsonized live S. epidermidis for 1 hour significantly increased the production of reactive oxygen species, the gene expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-10, as well as the secretion of TNF-α, demonstrating the ability of the cells to elicit a response and actively phagocytose prey. In addition, cells cultured on the smooth gold and the nanostructured gold displayed a different adhesion pattern and a more rapid oxidative burst than those cultured on polystyrene upon stimulation. We conclude that S. epidermidis decreased its viability initially when adhering to nanostructured surfaces compared with smooth gold surfaces, especially in the bacterial cell layers closest to the surface. In contrast, material surface properties neither strongly promoted nor attenuated the activity of monocytes when exposed to zymosan particles or S. epidermidis.

In vivo evaluation of noble metal coatings.

A nanotopographic noble metal (Ag, Au, Pd) coating has been applied on commercial urinary catheters and used in more than 80,000 patients, with good clinical results. We have previously evaluated the biocompatibility of different variations of this coating, showing high cellular viability and function in vitro. However, the reasons for good clinical and preclinical behavior are not known. This in vivo study aimed to investigate the soft tissue peri-implant reaction to five coatings with systematically altered noble metal ratios after 1, 3, and 21 days of implantation in rats. The results show that coatings of silver only, or silver with medium amounts of gold and low-medium palladium content were superior to other tested coatings. Such surfaces were during the first days after implantation associated with a decreased recruitment of inflammatory cells to implant close exudates, a lower percentage of neutrophils, higher cell viability, and lower production of monocyte chemoattractant protein-1 (MCP-1), compared to the other coatings and uncoated silicone (PDMS) control. In contrast, the addition of higher concentrations of gold and palladium to silver induced a thicker soft tissue capsule. Coatings with high concentration of palladium induced the thickest fibrouscapsule after 21 days of implantation. The study demonstrates that by varying the noble metal ratio at implant surfaces it is possible to modulate inflammation and fibrosis in soft tissue.

The influence of bone type on the gene expression in normal bone and at the bone-implant interface: experiments in animal model.

BACKGROUND Studies on the biological processes in different bone types and the reaction of different bone types to biomaterials are often hindered because of the difficulties in sampling procedures and lack of sensitive techniques. PURPOSE The purpose was to assess the suitability of quantitative polymerase chain reaction (qPCR) for investigation of the biological differences between cortical and trabecular bone types and their responses to biomaterials. MATERIALS AND METHODS Gene expression of selected markers in rat bone samples from different locations was evaluated. Samples were harvested by trephines from the trabecular femoral epiphysis, cortico-trabecular proximal tibial metaphysic, and the cortical distal tibial metaphysis. Gene expression was also evaluated at the surfaces of anodically oxidized implants retrieved from cortical and trabecular sites after 3 days of implantation. mRNA in the bone samples and in the tissue associated with the implant surfaces was extracted and quantified using qPCR. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), alkaline phosphatase (ALP), osteocalcin (OC), tartrate-resistant acid phosphatase (TRAP), cathepsin K (CATK), and 18S ribosomal subunits (18S) were analyzed. RESULTS In the bone samples, higher expression of ALP, OC, TRAP, and CATK was found in femoral epiphysis compared to proximal or distal tibial metaphysis, indicating a higher turnover in the trabecular bone. On the other hand, TNF-α and IL-1β showed higher expression in both tibia sites compared with the femur site, which suggests higher inflammatory potential in the cortical bone. In response to the oxidized implants trabecular bone expressed a higher level of IL-1β, whereas the implants in cortical bone were associated with higher expression of ALP and OC. CONCLUSION There are biological differences between cortical and trabecular bone types, both in the normal steady-state condition and in response to biomaterials. Such differences can be characterized and discriminated quantitatively using a sensitive technique such as qPCR.

A novel soft tissue model for biomaterial-associated infection and inflammation - Bacteriological, morphological and molecular observations

Infection constitutes a major risk for implant failure, but the reasons why biomaterial sites are more vulnerable than normal tissue are not fully elucidated. In this study, a soft tissue infection model was developed, allowing the analysis of cellular and molecular responses in each of the sub-compartments of the implant-tissue interface (on the implant surface, in the surrounding exudate and in the tissue). Smooth and nanostructured titanium disks with or without noble metal chemistry (silver, gold, palladium), and sham sites, were inoculated with Staphylococcus epidermidis and analysed with respect to number of viable bacteria, number, viability and gene expression of host cells, and using different morphological techniques after 4 h, 24 h and 72 h. Non-infected rats were controls. Results showed a transient inflammatory response at control sites, whereas bacterial administration resulted in higher recruitment of inflammatory cells (mainly polymorphonuclear), higher, continuous cell death and higher gene expression of tumour necrosis factor-alpha, interleukin-6, interleukin-8, Toll-like receptor 2 and elastase. At all time points, S. epidermidis was predominantly located in the interface zone, extra- and intracellularly, and lower levels were detected on the implants compared with surrounding exudate. This model allows detailed analysis of early events in inflammation and infection associated to biomaterials in vivo leading to insights into host defence mechanisms in biomaterial-associated infections.

Synthesis of 2′-([1,2,3]Triazol-1-yl)-2′-deoxyadenosines

A reliable and efficient protocol for the synthesis of 2 ′-([1,2,3]triazol-1-yl)-2 ′-deoxyadenosine derivatives from vidarabine is presented. Vidarabine was converted to 2′-azido-2′-deoxy-3′,5-O-(tetraisopropyldisiloxane-1,3-diyl)-adenosine. This azide was used as the starting material for the CuI-catalyzed parallel synthesis of 1,2,3-triazoles using a variety of alkynes. The reactions proceeded in good yield and gave almost exclusively the 1,4-disubstituted 1,2,3-triazoles.

Staphylococcal biofilm gene expression on biomaterials — A methodological study

The combination of increased healthcare access, universal aging, and infallible therapy demands, synergistically drive the need for the development of biomaterial technologies that mitigate the challenge of biomaterial-associated infections (BAI). Staphylococcus epidermidis and Staphylococcus aureus account for the majority of BAI due to their ability to accumulate in adherent multilayered biofilm. This investigation details the development of gene expression assays to evaluate the genetic processes of attachment, accumulation, maturation, and dispersal phases of biofilms on biomaterials in vitro, while abiding by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. The biofilm formation of S. epidermidis on polyurethane (PU) central venous catheters and S. aureus on machined titanium (Ti) was examined in terms of gene expression at early and late time points. The results provided insight into how each stage of biofilm formation is orchestrated over time on these biomaterials in vitro. Furthermore, the results suggested that mechanical RNA extraction, organic solvents, elimination of genomic DNA, and preamplification are advisable strategies to implement for biofilm gene expression analysis. It is concluded that this method can be employed for the assessment of biofilm-biomaterial interactions at the molecular level.