Nanostructured materials for inhibition of bacterial adhesion in orthopedic implants: a minireview.

Orthopedic implants may fail owing to different reasons: poor osseointegration at the tissue-implant interface, generation of wear debris, stress and strain imbalance between implant and surrounding tissues, and infections. To ensure success in orthopedics, implant materials must not evoke an undesirable inflammatory response, they must be habitable by bone-forming cells (favoring adhesion of osteoblasts), hinder formation of soft connective tissue (hindering adhesion of fibroblasts), and be anti-infective (discouraging bacterial adhesion). Recent studies have suggested that nanophase materials have a better efficacy as bone implants in favoring osseointegration compared to conventional orthopedic implant materials. This minireview discusses studies on nanophase materials as bone implants, focusing on the effect of these materials in inhibiting bacterial adhesion for the prevention of implant infections.

The selection of appropriate bacterial strains in preclinical evaluation of infection-resistant biomaterials.

Implant-related infections are broadly recognized as one of the most serious and devastating complications associated with the use of biomaterials in medical practice. The growing interest and need for the development of implant materials with reduced susceptibility to microbial colonization and biofilm formation has necessitated the development of a series of in vitro and in vivo models for evaluation and preclinical testing. Current technologies provide these investigations with an ample choice of qualitative and quantitative techniques for an accurate assessment of the bioactivity and anti-infective efficacy of any new compound or device. These tests are typically performed using a reference bacterial strain designated as the test or reference strain. Recent molecular epidemiological studies have identified the complex clonal nature of most prevalent etiological agents implicated in implant-associated infections. New information which is continually emerging on the identity and the characteristics of both sporadic and epidemic clones must be considered when selecting a reference. A new emerging requirement is that the strain should be representative of the clones causing clinically relevant infections; they should, therefore, belong to the most prevalent epidemic clones rather than to sporadic ones, which may occur in only 1 out of 200 infections or even fewer. The correct choice of reference strain for preclinical tests is of crucial importance for the clinical significance of the achieved results. In this paper we report our experience and recommendations regarding this issue.

Prevalence of genes encoding for staphylococcal leukocidal toxins among clinical isolates of Staphylococcus aureus from implant orthopedic infections.

Staphylococcus aureus has emerged as a major cause of implant infections. It is known that it is able to produce several toxins that contribute to its armory of virulent weapons, but there are still no data on their prevalence among isolates recovered from biomaterial-centered infections. In this study, 200 Staphylococcus aureus isolates from infections related to different types of orthopedic implants (hip and knee arthroprostheses, internal and external fixation devices) were tested by polymerase chain reaction for the prevalence of genes encoding for leukotoxins. Although almost all isolates were positive for the ã-hemolysin gene (99%), none was positive for lukM. The leukotoxin genes lukE/lukD were found in 67% of isolates. The presence of lukE/lukD was significantly associated with that of Accessory Gene Regulatory locus agr II. The lukE/lukD-positive isolates were significantly more prevalent in the staphylococcal isolates from knee arthroprostheses than in the isolates from the other implant types. The genes encoding Panton-Valentine leukocidin components were detected in only one isolate that, curiously enough, was taken solely from a knee arthroprosthesis infection.

Innovative methods of rapid bacterial quantification and applicability in diagnostics and in implant materials assessment.

In recent years, a variety of new technologies have been proposed that allow rapid qualitative and quantitative microbiological analyses. In this paper we discuss the urgent needs for reliable and rapid microbiological analytical techniques in different applicative fields involving the research, production and medical application of implant materials, and the potential benefits derived from the use of new methods for rapid bacterial quantification. Current compendial methods are easy to perform and have gained confidence over their long period of use, but the supplemental use of new technologies could represent real breakthroughs whenever sensitive and rapid responses are urgently required and not met by the tests currently in use. Overall, the new microbiological methods require critical evaluation depending on their specific type of application and they may still not be thought of as totally substitutive, but they certainly exhibit considerable potential for different areas of biomaterials, as well as for advanced therapy medicinal and tissue engineering treatments.

Study of Staphylococcus aureus adhesion on a novel nanostructured surface by chemiluminometry.

In recent years the progress in the field of nanotechnologies has offered new possibilities to control the superficial features of implant materials down to a nanoscale level. Several studies have therefore tried to explore the effects of nanostructured biomaterial surfaces on the behavior of eukaryotic cells. However, nanotopography could exert an influence also on the behavior of prokaryotic cells, with relevant implications concerning the susceptibility of implant surfaces to infection. Aim of this study was to examine the behavior of Staphylococcus aureus on polyethylene terephthalate (PET) surfaces either cylindrically nanostructured (PET-N) or flat ion-etched (PET-F), and on tissue culture-grade polystyrene (PS). Microbial adherence was assessed by chemiluminometry under 4 different conditions: (a) bacteria suspended in MEM medium, (b) bacteria in MEM supplemented with 10% fetal bovine serum (FBS), (c) test surfaces preconditioned in FBS, and (d) post-exposure of colonised surfaces to serum-supplemented MEM. Under all circumstances, PET-F and PET-N specimens showed identical bacterial adhesion properties. In the absence of serum, all 3 test materials showed a very high adhesivity to microbial cells and both PET surfaces exhibited greater adhesion than PS. On the contrary, the presence of 10% serum in solution significantly affected cell behavior: the number of microbial cells on all surfaces was drastically reduced, and the adhesion properties of PET surfaces with respect to PS were reversed, with PET being less adhesive. Overall, the specific cylindrical nanostructures created on PET did not significantly influence microbial behavior. Ongoing studies are verifying whether other nanotopographies with different geometry could have more substantial effects.

Emerging Staphylococcus species as new pathogens in implant infections.

The vast use of prosthetic materials in medicine over the last decades has been accompanied by the appearance of new opportunistic pathogens previously considered incapable of causing infections with significant morbidity and/or mortality. In this regard, the genus Staphylococcus enlisting numerous species usually characterized by a saprophytic habit covers a special role. Apart from Staphylococcus aureus and Staphylococcus epidermidis, well known for their large prevalence in implant-related infections, a number of further staphylococcal species are progressively being indicated for their pathogenic potential. The increasing attention on these opportunistic bacteria is due to an ever growing number of clinical reports, which is also deriving from a more accurate identification of these species with currently available techniques. This synopsis intends to offer an overview on recently emerging coagulase-negative staphylococci (CoNS) as well as coagulase-positive/-variable staphylococci exhibiting distinct traits of virulence, pathogenicity, and epidemiologic impact depending among others on the medical field, the type of prosthetic device and its anatomic location.

Prevalence and antibiotic resistance of 15 minor staphylococcal species colonizing orthopedic implants.

Several species belonging to Staphylococcus genus (non Sau/ non Sep species) exhibit increasing abilities as opportunistic pathogens in colonisation of periprosthesis tissues. Here we report on antibiotic resistance of 193 strains, belonging to non Sau/ non Sep species, consecutively collected from orthopedic implant infections in a period of about 40 months. The 193 strains (representing 17% of all staphylococci isolated) were analysed for their antibiotic resistance to 16 different drugs. Five species turned out more prevalent, ranging from 1 to 5%: S. hominis (4.2%), S. haemolyticus (3.7%), S. capitis (2.7%), S. warneri (2.6%), and S. cohnii (1.6%). Among these, the prevalence of antibiotic resistance to penicillins was similar, ranging from 51% to 66%. Conversely, significant differences were observed for all the remaining antibiotics. For S. haemolyticus the resistances to oxacillin and imipenem, the four aminoglycosides and erythromycin were at least twice that of the other three species which were compared. S. warneri was on the contrary the species with the lowest occurrence of resistant strains. Ten species appeared only rarely at the infection sites: S. lugdunensis, S. caprae, S. equorum, S. intermedius, S. xylosus, S. simulans, S. saprophyticus, S. pasteuri, S. sciuri, and S. schleiferi. The behaviours of these species, often resistant to penicillins, were individually analysed. Differences in both the frequencies and the panels of antibiotic resistances observed among the non Sau/ non Sep species: i) suggest that horizontal spreading of resistance factors, if acting, was not sufficient per se to level their bio-diversities; ii) highlight and confirm the worrisome appearance within the Staphylococcus genus of emerging ""new pathogens"", not homogeneous for their virulence and antibiotic resistance prevalence, which deserve to be recognised and treated individually.

Virulence factors in enterococcal infections of orthopedic devices.

Enterococci are opportunistic pathogens which today represent one of the leading causes of nosocomial infections. We have examined a collection of 52 Enterococcus faecalis isolated from orthopedic infections to determine if they were characterized by a specific pattern of virulence factors. The isolates were evaluated for biofilm formation, presence of genes coding the enterococcal surface protein (esp) and gelatinase (gelE), as well as for gelatinase production. While the rate of esp-positive isolates was comparable to that found among strains from other clinical sources, we found a significantly higher rate of strong biofilm formers and gelatinase producers. Particularly high was the rate of gelE-carrying strains expressing the gene. Data suggest that these two factors in particular may play an important role in enterococcal infections associated with biomaterials.

Automated ribotyping to distinguish the different non Sau/ non Sep staphylococcal emerging pathogens in orthopedic implant infections.

Several species belonging to Staphylococcus genus, other than Staphylococcus aureus and Staphylococcus epidermidis (non Sau/ non Sep species), exhibit increasing abilities as opportunistic pathogens in the colonisation of periprosthetic tissues. Consequently, the availability of means for accurate identification is crucial to assess the pathogenic characteristics and to clarify clinical relevance of the individual species. Here, 146 clinical staphylococcal isolates belonging to non Sau/ non Sep species from prosthesis-associated orthopedic infections were analyzed by conventional enzymatic galleries and by automated ribotyping. Twelve different species were recognised: S. capitis, S. caprae, S. cohnii, S. equorum, S. haemolyticus, S. hominis, S. lugdunensis, S. pasteuri, S. sciuri, S. simulans, S. warneri, S. xylosus. Ribotype identifications were compared with the phenotypes obtained by the Api 20 Staph system and/or ID 32 Staph system. ID 32 Staph profiles were more consistent with ribotyping results than Api Staph profiles. Across the different staphylococcal species investigated, correct identifications with Api Staph were 45%, while with ID 32 Staph they were 59%. It has, however, to be mentioned that ID 32 Staph was mostly applied to discriminate unmatched ribotyping and Api Staph identifications, thus to a subpopulation of strains with ""atypical"" metabolic profile. Automated ribotyping provided a correct identification for 91% of the isolates. These results confirm automated ribotyping as a convenient rapid technique, still subject to improvements, which will accurately and rapidly recognise the newly emerging staphylococcal pathogens in implant-related orthopedic infections.

A glance at the role of exotoxins in opportunistic bacterial infections.

The production and the mechanism of action of exotoxins from Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa are presented. The attack to the immune host's defenses is the main virulence factor of opportunistic bacteria in implant infections, favoring the invasion and colonization of compromised periprosthesis tissues.

Etiology of implant orthopedic infections: a survey on 1027 clinical isolates.

In spite of the recent achievements derived from modern protocols of prophylaxis, orthopedic surgical infections still remain unacceptably frequent, especially in light of the often devastating outcomes of septic complications. The spectrum and the prevalence of the bacteria most frequently involved in orthopedic infections are here explored, with particular reference to those infections associated to implant biomaterials, which were grouped based on device typology. During a 30 months period (from September 2000 to April 2003), 1027 microbial strains were consecutively isolated from 699 patients undergoing revision surgery at the Rizzoli Orthopedic Institute. 775 (75.5%) of all these microorganisms were identified as belonging to the Staphylococcus genus, 82 (8%) to the Enterobacteriaceae family, 75 (7.3%) to the Pseudomonas genus, 54 (5.3%) to the Enterococcus genus and 20 (1.9%) to the Streptococcus genus. While confirming the importance of staphylococci as the most diffuse cause of infection, our data indicate an unexpectedly high prevalence of S. epidermidis on infected hip and knee arthroprostheses, respectively of 42% and 44%. The spectrum of bacteria infecting either internal or external fracture fixation devices appears to differ from that of hip and knee arthroprostheses and more closely resembles that of infections non-associated to medical devices, being characterized by a relatively higher prevalence of Staphylococcus aureus (over 40%) and Pseudomonas aeruginosa. Enterobacteriaceae and members of the Streptococcus and Corynebacterium genera are frequently associated with implants in which surgical incisions were made near the perineum, determining a completely altered spectrum.

Implant infection and infection resistant materials: a mini review.

Implant infection is an aggressive, often irreducible post-surgical infection. It remains the primary cause of implant failure. Bacterial contamination during surgery and subsequent adhesion onto biomaterial surface of opportunistic microorganisms, such as staphylococcal species, exopolysaccharidic slimes or specific adhesins, initiates the implant infection. Pathogenesis of periprosthestic infection is the focus of studies aimed at developing infection resistant materials.

Antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis clinical isolates from implant orthopedic infections.

In the last decade the rising phenomenon of resistance to most common antibiotic drugs among staphylococcal clinical isolates has been a reason for serious concern and alarm. The present study investigated the prevalence of antibiotic resistance within a large microbial collection including 530 clinical strains of S. aureus and 408 strains of S. epidermidis to a panel of 16 different drugs. All strains were isolated from orthopedic infections, either associated or non-associated with implant materials. Interestingly, our data show that the profile of the prevalence of antibiotic resistance within the two species of pathogens is extremely similar for the vast majority of the drugs screened. The only statistically significant variations in prevalence concerned, in order of relevance, the following 5 out of 16 antibiotics: sulfamethoxazole (in combination with trimethoprim), erythromycin, and, to a lesser extent, oxacillin, imipenem, and clindamycin. In the case of Staphylococcus aureus, the isolates associated to implant materials were found more frequently resistant to all 4 aminoglycosides screened as well as to ciprofloxacin.

No genotoxicity of a new nickel-free stainless steel.

Stainless steel is a metallic alloy largely employed in orthopedics, maxillofacial surgery and orthodontic therapy. However, the presence in its composition of a high quantity of nickel, an agent known to trigger toxic, allergic and cancerogenous responses in humans, is cause of some concern. In this study, we have investigated the in vitro mutagenicity and genotoxicity of a new nickel-free stainless steel, namely P558, in comparison to the conventional stainless steel AISI 316L. The cytogenetic effects were evaluated by studying the frequency of Sister Chromatid Exchanges (SCE) and chromosomal aberrations. Ames test was performed to detect the mutagenic activity. Both P558 and AISI 316L did not cause any significant increase in the average number of SCE and in chromosomal aberrations, either with or without metabolic activation. Furthermore, the Ames test showed that the extracts of both P558 and of AISI 316L are not mutagenic. Overall, these findings prove that P558 is devoid of genotoxicity and mutagenicity. The present results, together with other previous interesting observations that P558 promotes osseointegration, suggest that this new nickel-free stainless steel can represent a better alternative to other conventional steel alloys.

In vitro effects on MG63 osteoblast-like cells following contact with two roughness-differing fluorohydroxyapatite-coated titanium alloys.

The chances of integration between an implant and the surrounding bone tissue depend on the surface characteristics of the implant itself. Particularly, chemical composition and surface roughness of the material have emerged as crucial factors in affecting the behaviour of cells in contact with the material. Among various surfaces, calcium phosphate coatings seem to favour a rapid initial integration, but their dissolution by extracellular fluids raises some concern about the long-term stability at the bone-implant interface. Fluorinated apatites are known to be more stable than other ceramic coatings, but, at present, little is known on their effects on human cells. In this study, MG63 osteoblast-like cells were seeded onto two fluorohydroxyapatite (FHA)-coated titanium alloy (Ti6Al4V) materials differing in roughness, respectively, LR-FHA (Ra = 5.6 microm) and HR-FHA (Ra = 21.2 microm). Quantification of the cells in contact with the FHA-coated materials by conventional methods involved some technical difficulties, on which we report. Only the indirect esteem by the measure of total content of proteins and a procedure based on cell count, following a double enzymatic treatment to detach the cells, offered plain results, indicating no significant differences between cellular growth in contact with test materials and with plastic control. Differentiation and functionality of the cells were comparatively evaluated by analysis of alkaline phosphatase activity and osteocalcin production. As far as osteocalcin release is concerned, only slight variations were detected on FHA-coated materials in comparison with the control. Both types of coatings showed a significant increase in alkaline phosphatase activity with respect to the control, the roughest surface exhibiting a more prolonged effect on the time.

Congo red agar plate method: improved accuracy and new extended application to Staphylococcus aureus.

In the last decade an increasing number of research studies have focused on the role of slime formation in Staphylococcus epidermidis and, more recently, also in S. aureus. In this context, much attention is being paid to evaluating the prevalence of slime production among bacteria strains isolated from clinical infections in an attempt to assess the role and the diagnostic value of this well recognised virulence marker. Such types of investigations require reliable techniques to identify slime producing strains. For years, even though based on a subjective chromatic evaluation, the Congo red agar plate (CRA) represented a reference phenotypic test for S. epidermidis. Only with the new introduction of PCR-based techniques, able to specifically identify the genes necessary for slime production, did the accurate genetic classification of slime producing bacteria become possible. In the present investigation, a comparison with new PCR methods confirmed the validity of the classic CRA test, implemented with minor refinements. Thanks to a few modifications it was also possible to adapt and extend the CRA test, making it also suitable to screen S. aureus strains.

Cartilage tissue engineering with novel nonwoven structured biomaterial based on hyaluronic acid benzyl ester.

The aim of this study was to investigate the possibility of using the benzyl ester of hyaluronic acid (HYAFF 11), a recently developed semisynthetic resorbable material, as a scaffold for the culture of human nasoseptal chondrocytes in tissue-engineering procedures of cartilage reconstruction. Different techniques such as immunohistochemistry, scanning electron microscopy, and confocal laser scanning microscopy were used to study the behavior, morphology, and phenotype expression of the chondrocytes, which were initially expanded and then seeded on the material. The nonwoven cell carrier allowed good viability and adhesivity of the cells without any surface treatment with additional substances. Furthermore, the cultured cells expressed cartilage-specific collagen type II, indicating that they were able to redifferentiate within the scaffold of HYAFF 11 and were able to retain a chondrocyte phenotype even after a long period of in vitro conditions. Nevertheless, the expression of collagen type I, which was produced by dedifferentiated or incompletely redifferentiated chondrocytes, was noticeable. Additional data were obtained by subcutaneous implantation of samples seeded with human cells in the in vivo model of the athymic nude mouse. The results after 1 month revealed the development of tissue similar to hyaline cartilage. This study is promising for the use of this scaffold for tissue engineering of cartilage replacements.

Semisynthetic resorbable materials from hyaluronan esterification.

In recent years, research on new, biocompatible, degradable materials has seen the development of a series of modified natural polymers. Among these, a new class of materials consisting of different hyaluronan derivatives promises to be useful in a whole range of clinical applications thanks to their varied biological properties. These new materials are obtained by chemical modification of purified hyaluronan consisting of the partial or total esterification of the carboxyl groups of this natural polymer. This review on the properties of the new materials reports some of their biocompatibility and characterization aspects based on findings from studies conducted on the ethyl and benzyl hyaluronan esters, two representative members of this new class of compounds, and is intended to arouse interest in the potential of other, as yet unexplored derivatives. From the results of a number of investigations, the various derivatives appear to possess different physico-chemical properties, especially as far as the degree of hydration and polymer stability are concerned. In addition, the type of esterification and extent of chemical esterification of hyaluronan considerably affects the biological properties of these materials, offering a range of polymers either favouring or, conversely, inhibiting the adhesion of certain types of cell.

Quantitative assessment of the tissue response to films of hyaluronan derivatives.

The aim of this study was to evaluate the in vivo response following implantation into a rat model of three innovative hyaluronan derivatives for clinical use: HYAFF 7, HYAFF 11 and HYAFF 11p75 (respectively, the 100% ethyl ester, 100% and 75% benzyl esters). The tissue reaction evoked by films of these new biomaterials implanted into the dorsolumbar musculature of rats was assessed quantitatively using a well established technique based upon an image analysis system. The number of inflammatory cells present and the patterns of cell distribution around the implant up to a distance of 642 microns were examined at different time periods after implantation. Since a well-delineated tissue-material interface was needed for this type of investigation, it was not possible to apply image analysis to sections once dissolution of the implanted materials had begun. Films of both the total esters, HYAFF 7 and HYAFF 11, were found to undergo a slow dissolution process and, after a month, films of these materials were still present at the site of implantation. Differences in response to the two materials were observed only during the first two weeks, particularly with respect to neutrophil distribution and total cellularity. HYAFF 7 was found to be more reactive, with higher numbers of neutrophils near the surface of the implant than HYAFF 11. Thereafter, the differences between the two materials were minimal and owing mainly to a faster dissolution of HYAFF 7 films. After 3 and 5 months, considerable degradation of films of both total esters had occurred. Significant quantities of material appeared inside numerous macrophages with an ED1-positive phenotype. Only a very thin layer of fibrous connective tissue, indicative of low reactivity, was found to surround the site of implantation, separating the dissolved material and the phagocytic cells from healthy muscular tissue. ED2-positive macrophages were primarily confined within the lining connective tissue. The partial benzyl ester, HYAFF 11p75, showed a different behaviour. In fact, evidence of film dissolution was already present a week after the implantation. After two weeks, the implanted films were completely dissolved and numerous ED1-positive macrophages phagocytosing the material were observed at the site of implantation. Therefore, in agreement with previous in vitro studies, which showed a greater susceptibility to degradation of hyaluronan derivatives with lower percentage of esterification, HYAFF 11p75 underwent resorption faster than the corresponding total ester.

Biodegradation of hyaluronic acid derivatives by hyaluronidase.

Hyaluronic acid (salt) (HA) has been chemically modified as a biomaterial for medical applications such as controlled drug release matrices, nerve guides and wound dressings. A series of HA derivatives, which include different ester types and different degrees of esterification, have been used to investigate the stability of these materials in testicular hyaluronidase. Gel permeation chromatography and capillary viscometer have been employed to determine the size of the molecules, the former used for the water insoluble derivatives that dissolve in dimethyl sulphoxide, the latter for the water soluble samples. The preliminary experimental results indicated that the molecular weight of fully esterified hyaluronic acid (both ethyl and benzyl esters) did not decrease after treatment in the enzyme for 7 and 14 days while the water soluble partially esterified HA were degraded by the enzyme producing a sharp reduction of viscosity within minutes. These observations tend to suggest that the carboxylic groups in the beta-glucoronic acid unit are the activation centre of this enzyme and the total blockage of these groups can restrict the cleavage of beta (1-->4) glycoside bonds by this enzyme.