Margarita Trobos

Commercially pure titanium (cp-Ti) versus titanium alloy (Ti6Al4V) materials as bone anchored implants - Is one truly better than the other?

Commercially pure titanium (cp-Ti) and titanium alloys (typically Ti6Al4V) display excellent corrosion resistance and biocompatibility. Although the chemical composition and topography are considered important, the mechanical properties of the material and the loading conditions in the host have, conventionally, influenced material selection for different clinical applications: predominantly Ti6Al4V in orthopaedics while cp-Ti in dentistry. This paper attempts to address three important questions: (i) To what extent do the surface properties differ when cp-Ti and Ti6Al4V materials are manufactured with the same processing technique?, (ii) Does bone tissue respond differently to the two materials, and (iii) Do bacteria responsible for causing biomaterial-associated infections respond differently to the two materials? It is concluded that: (i) Machined cp-Ti and Ti6Al4V exhibit similar surface morphology, topography, phase composition and chemistry, (ii) Under experimental conditions, cp-Ti and Ti6Al4V demonstrate similar osseointegration and biomechanical anchorage, and (iii) Experiments in vitro fail to disclose differences between cp-Ti and Ti6Al4V to harbour Staphylococcus epidermidis growth. No clinical comparative studies exist which could determine if long-term, clinical differences exist between the two types of bulk materials. It is debatable whether cp-Ti or Ti6Al4V exhibit superiority over the other, and further comparative studies, particularly in a clinical setting, are required.

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.

Bacteria‐material surface interactions: methodological development for the assessment of implant surface induced antibacterial effects

The choice of material for implanted prostheses is of great importance concerning bacterial colonization and biofilm formation. Consequently, methods to investigate bacterial behavior are needed in order to develop new infection resistant surfaces. In this study, different methodological setups were used to evaluate the antimicrobial effect of photocatalytic titanium oxide and silver surfaces. Biofilm formation and eradication under static and dynamic culture conditions were studied with the use of the following analytical techniques: viable colony-forming unit (CFU) counting, imprinting, fluorescence, and bioluminescence. The present study demonstrates that different methods are needed in order to evaluate the prophylactic and treatment effects on planktonic and biofilm bacteria and to assess the antimicrobial effect of different surface treatments/coatings. Choosing the right antibacterial testing model for the specific application is also of great importance. Both in situ approaches and indirect methods provide valuable complementary information.

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.

Biofilm formation and antimicrobial susceptibility of staphylococci and enterococci from osteomyelitis associated with percutaneous orthopaedic implants

Staphylococci and enterococci account for most deep infections associated with bone-anchored percutaneous implants for amputation treatment. Implant-associated infections are difficult to treat; therefore, it is important to investigate if these infections have a biofilm origin and to determine the biofilm antimicrobial susceptibility to improve treatment strategies. The aims were: (i) to test a novel combination of the Calgary biofilm device and a custom-made susceptibility MIC plate (Sensititre® ), (ii) to determine the biofilm formation and antimicrobial resistance in clinical isolates causing implant-associated osteomyelitis, and (iii) to describe the associated clinical outcome. Enterococci and staphylococci were characterized by microtitre plate assay, Congo Red Agar plate test, and PCR. Biofilm susceptibility to 10 antimicrobials and its relationship to treatment outcomes were determined. The majority of the strains produced biofilm in vitro showing inter- and intraspecies differences. Biofilms showed a significantly increased antimicrobial resistance compared with their planktonic counterparts. Slime-producing strains tolerated significantly higher antimicrobial concentrations compared with non-producers. All seven staphylococcal strains carried ica genes, but two did not produce slime. The degree of biofilm formation and up-regulated antibiotic resistance may translate into a variable risk of treatment failure. This new method set-up allows for the reproducible determination of minimum biofilm eradication concentration of antimicrobial agents, which may guide future antimicrobial treatment decisions in orthopaedic implant-associated infection.

The clinical, radiological, microbiological, and molecular profile of the skin-penetration site of transfemoral amputees treated with bone-anchored prostheses

Abstract The breach of the skin barrier is a critical issue associated with the treatment of individuals with transfemoral amputation (TFA) using osseointegrated, percutaneous titanium implants. Thirty TFA patients scheduled for abutment exchange or removal were consecutively enrolled. The aims were to determine the macroscopic skin signs, the presence of bacteria and the gene expression in abutment‐adherent cells and to conduct correlative and comparative analyses between the different parameters. Redness and a granulation ring were present in 47% of the patients. Bacteria were detected in 27/30 patients, commonly in the bone canal. Staphylococcus aureus, coagulase‐negative staphylococci, streptococci, and Enterococcus faecalis were the most common. A positive correlation was found between TNF‐α expression and the detection of S. aureus. Staphylococcus aureus together with other bacterial species revealed a positive relationship with MMP‐8 expression. A negative correlation was demonstrated between the length of the residual femur bone and the detection of a granulation ring and E. faecalis. A positive correlation was revealed between fixture loosening and pain and the radiological detection of endosteal bone resorption. Fixture loosening was also correlated with the reduced expression of interleukin‐10 and osteocalcin. It is concluded that several relationships exist between clinical, radiological, microbiological, and molecular assessments of the percutaneous area of TFAs. Further long term studies on larger patient cohorts are required to determine the precise cause‐effect relationships and unravel the role of host‐bacteria interactions in the skin, bone canal and on the abutment for the longevity of percutaneous implants as treatment of TFA.

Effect of load on the bone around bone-anchored amputation prostheses

Osseointegrated transfemoral amputation prostheses have proven successful as an alternative method to the conventional socket‐type prostheses. The method improves prosthetic use and thus increases the demands imposed on the bone‐implant system. The hypothesis of the present study was that the loads applied to the bone‐anchored implant system of amputees would result in locations of high stress and strain transfer to the bone tissue and thus contribute to complications such as unfavourable bone remodeling and/or elevated inflammatory response and/or compromised sealing function at the tissue‐abutment interface. In the study, site‐specific loading measurements were made on amputees and used as input data in finite element analyses to predict the stress and strain distribution in the bone tissue. Furthermore, a tissue sample retrieved from a patient undergoing implant revision was characterized in order to evaluate the long‐term tissue response around the abutment. Within the limit of the evaluated bone properties in the present experiments, it is concluded that the loads applied to the implant system may compromise the sealing function between the bone and the abutment, contributing to resorption of the bone in direct contact with the abutment at the most distal end. This was supported by observations in the retrieved clinical sample of bone resorption and the formation of a soft tissue lining along the abutment interface.

In vitro evaluation of barrier function against oral bacteria of dense and expanded polytetrafluoroethylene (PTFE) membranes for guided bone regeneration

AIM This study evaluates biofilm formation and barrier function against Streptococcus oralis of nonresorbable polytetrafluoroethylene (PTFE) guided bone regeneration membranes having expanded (e-PTFE) and dense (d-PTFE) microstructure. MATERIALS AND METHODS Three e-PTFE membranes of varying openness, one d-PTFE membrane, and commercially pure titanium discs were evaluated. All e-PTFE membranes consisted of PTFE nodes interconnected by fibrils. The d-PTFE membrane was fibril-free, with large evenly spaced indentations. The surfaces were challenged with S. oralis and incubated statically for 2-48h. Bacterial colonization, viability, and penetration were evaluated. RESULTS S. oralis numbers increased over time on all surfaces, as observed using scanning electron microscopy, while cell viability decreased, as measured by colony forming unit (CFU) counting. At 24h and 48h, biofilms on d-PTFE were more mature and thicker (tower formations) than on e-PTFE, where fewer layers of cells were distributed mainly horizontally. Biofilms accumulated preferentially within d-PTFE membrane indentations. At 48h, greater biofilm biomass and number of viable S. oralis were found on d-PTFE compared to e-PTFE membranes. All membranes were impermeable to S. oralis cells. CONCLUSIONS All PTFE membranes were effective barriers against bacterial passage in vitro. However, d-PTFE favored S. oralis biofilm formation.

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.