Aims

Electromagnetic induction heating has demonstrated in vitro antibacterial efficacy over biofilms on metallic biomaterials, although no in vivo studies have been published. Assessment of side effects, including thermal necrosis of adjacent tissue, would determine transferability into clinical practice. Our goal was to assess bone necrosis and antibacterial efficacy of induction heating on biofilm-infected implants in an in vivo setting.

Aim

To evaluate the bacterial counts of sonicatied implants in patients with osteoarticular infections. Various studies have demostrated the usefulness of sonication of retrieved implants in order to provide an accurate microbiological diagnosis. Although cutoff values for original sonicate counts have been established, the use of centrifugation may influence these values

In the past decades, titanium-based biomaterials have been broadly used in maxillofacial and periodontology surgery. The main aetiological agents related to complications in this procedures are Porphyromonas gingivalis, a Gram-negative anaerobic bacteria that is also responsible for the development of chronic gingivitis, and Streptococcus oralis, a Gram positive facultative anaerobic bacteria. In previous studies, we have demonstrated that the fluorine doping of titanium-based alloys reduces bacterial adherence.

The aim of this study is to evaluate the bacterial adherence on fluorine-doped titanium (TiF) probes compared to chemical polishing titanium (Ti) probes.

The P. gingivalis ATCC 33277 and S. oralis ATCC 9811 adherence study was performed by introducing each probe in a well of 6-well plate with 5 ml containing 106 colony forming units (CFU/ml) in sterile 0.9% NaCl and was incubated 37°C 5% CO2 for 90 minutes, in anaerobiosis in the case of P. gingivalis. After incubation, samples were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Proportion of live and dead bacteria was calculated and studied by using ImageJ software. The experiments were performed in triplicate.

The statistical data were analyzed by nonparametric Wilcoxon test with a level of statistical significance of 0.05. Our results showed a significant (p<0.0053) 14.41% decrease of the adherence of P. gingivalis on TiF and an increase of 30% of dead cells. For S. oralis we did not get significant results.

In conclusion, TiF can be considered a promising approach to prevent and treat infections related to maxillofacial and periodontology surgery.

Prosthetic joint infections (PJI) occur infrequently, but they represent the most devastating complication with high morbidity and substantial cost. Staphylococcus aureus and coagulase-negative S. epidermidis are the most common infecting agents associated with PJI.

During the past decades, novel materials have been developed to improve osseointegration of implants. Recently has been demonstrated that by using nanosized hydroxyapatite (HA) coatings, since it combines nanoroughness and bone-like chemistry in a synergistic effect, it promotes better osseointegration when compared to uncoated metal implants. In a further step, due to the known bactericidal properties of fluor, the aim of this study is to evaluate the biofilm development on fluorohydroxyapatite (FHA) compared to HA.

Coatings were grown on stainless steel substrates by Pulsed Laser Deposition (PLD) technique using fluorohydroxyapatite targets of marine origin. A comprehensive physicochemical characterization of the coatings was performed using SEM, EDS, XPS and XRD. Biological in vitro tests using the pre-osteoblastic cell line (MC3T3-E1) demonstrated the non-cytotoxicity of FHA coatings, the healthy cell proliferation and their osteogenic activity.

The S. aureus 15981 (Valle et al.) and S. epidermidis ATCC 35984 strains adherence study was performed introducing each probe in a well of 96-well plate with 200 µl containing 106 colony forming units (CFU/mL) intryptic soy broth supplemented with 1% glucose and was incubated at 37°C 5% CO2 for 24 hours. After incubation, the medium was removed and three washes with 0.9% NaCl sterile saline were performed. The biofilm was disrupted by sonication at 50–60 Hz for 5 min. The CFU/cm2 was estimated by drop plate method. All of the experiments were performed in triplicate.

Statistical analysis was performed by non-parametric unilateral Wilcoxon”s test with a level of statistical significance of 0.05.

The results showed a significant (p=0.02475) 2.4-fold reduction in S. epidermidis biofilm formation on FHA (logUFC/cm2 = 6.87) compared to HA (logUFC/cm2 = 7.25); and also a significant (p=0.042) 3.7-fold reduction in S. aureus 15981 biofilm formation.

In conclusion, according our results FHA is a promising biomaterial that promotes osseointegration and decreases the staphylococcal biofilm that could avoid PJI. Further studies will be necessary.

Prosthetic joint infections (PJI) occur infrequently, but they represent the most devastating complication with high morbidity and substantial cost. Staphylococcus aureus and coagulase-negative S. epidermidis are the most commonly infecting agents associated with PJI. Nowadays, Gram-negative species like Escherichia coli and Pseudomonas aeruginosa are gaining relevance.

The use of TiO2 conical nanotubular doped with fluorine and phosphorous (FP-cNT) surfaces is an interesting approach to prevent surface bacterial colonization during surgery and favouring the osseointegration. Despite of there are serum markers related with PJI, to date there is described no biomaterial-related marker that allows detecting PJI. Here we describe the adherence and the bactericidal effect of FP-cNT and its capacity of marking the non-fermenting bacteria that have been in contact with it by Al. This metal is delivered by FP-cNT in non-toxic concentrations (between 25 and 29 ng/mL).

F-P-cNT layers on Ti6Al4V alloy were produced as described previously by our group. Ti6Al4V chemical polishing (CP) samples without nanostructure were used as control and produced as described previously.

S. aureus 15981, S. epidermidis ATCC 35984, E. coli ATCC 25922, and P. aeruginosa ATCC 27853 strains adherence study was performed using the protocol described by Kinnari et al. in 0.9% NaCl sterile saline with a 120 min incubation. After incubation, the samples were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Proportion of live and dead bacteria was calculated and studied by using ImageJ software. The experiments were performed in triplicate. The aluminum concentration was estimated in the supernatant after incubation and in the 0.22 µm filtered supernatant by atomic absorption in graphite furnace.

The statistical data were analyzed by nonparametric Kruskal-Walis test and by pairwise comparisons using the nonparametric unilateral Wilcoxon test with a level of statistical significance of p<0.05. The values are cited as medians.

Our results show that the bacterial adherence of all tested species significantly decreased on FP-cNT compared to CP except P. aeruginosa ATCC 27853: 19.8% for S. aureus 15981, 45.3% for S. epidermidis ATCC 35984 and 8.1% for E. coli ATCC 25922. The bacterial viability decreased 2-fold for S. aureus 15981, and 5-fold for S. epidemidis ATCC 35984, but increased 95% for P. aeruginosa ATCC 27853 and there no was variation for E. coli ATCC 25922 on FP-cNT compared to CP. Only supernatant P. aeruginosa ATCC 27853 shows significant Al detection after 120 min incubation (p<0.05).

In summary, F-P cNT is a promising biomaterial that besides favoring osseointegration and potential usefulness as drug carrier, present bactericidal, non-stick ability (at least for staphylococci and E. coli) and is able to mark P. aeruginosa with Al, which could be potentially monitored in serum and urine in patients with PJI.

Background

Staphylococcus aureus is a human pathogen involved in implant-related infections. In these diseases, biofilm production is the key pathogenic event, and it increases antibiotic resistance of the organism. Because this phenomenon, local delivery of antibiotics could allows reaching high concentrations in the infected tissue without the secondary effects linked to systemic administration. Here we report the use of a ceramic biomaterial (SBA-15) as a carrier of antibiotics in order to deliver them directly in the infected tissue.

Objectives

Implant-related infection is one of the most devastating complications in orthopaedic surgery. Many surface and/or material modifications have been developed in order to minimise this problem; however, most of the in vitro studies did not evaluate bacterial adhesion in the presence of eukaryotic cells, as stated by the ‘race for the surface’ theory. Moreover, the adherence of numerous clinical strains with different initial concentrations has not been studied.

Introduction

Prosthetic joint infections (PJI) occur infrequently, but due to its increased clinical use represent the most devastating complication with high morbidity and substantial cost. Staphylococcus aureus and coagulase-negative staphylococci are the most common infecting agents associated with PJI. A possible therapeutic approach could be the local antibiotic by fluoride-TiO2 nanostructured anodic layers in order to prevent surface colonisation during the early moments after surgery. Here we describe the first results of this model using two common antibiotics.

Prosthetic joint infections (PJI) occur infrequently, but they represent the most devastating complication with high morbidity and substantial cost. Staphylococcus aureus are the most common infecting agents associated with acute PJI, and also appear in some cases of delayed PJI¹. S. aureus biofilm development can be divided in two stages: adhesion and proliferation². To avoid PJI bacterial adhesion has to be decreased.

Hybrid organo-inorganic sol-gel coatings are proposed as a promising biomaterial improvement³. One of these compounds is a mixture of two organopolisiloxanes: 3-methacryloxypropyltrimethoxysilane (MAPTMS) and tetramethylorthosilicate (TMOS). The aim of this work was to evaluate bacterial adhesion on MAPTMS-TMOS coating compared to titanium parts made by powder metallurgy.

MAPTMS-TMOS sol-gel coating was produced using a molar ratio of 1:2 (MAPTMS:TMOS) and dispersed in ethanol. The sol-gel was deposited by dip-coating on titanium parts made by powder metallurgy followed by a thermal treatment at 120 ºC for 30 minutes⁴. Titanium parts without sol-gel coating were used as control.

S. aureus 15981 strain adherence study was performed using the protocol described by Kinnari et al.⁵ with 90 min incubation. After incubation, the samples were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Proportion of total adhered, live and dead bacteria was calculated and studied by using ImageJ software. The experiments were performed in triplicate.

The statistical data were analyzed by pairwise comparisons using the nonparametric Mann-Whitney test with a level of statistical significance of p<0.05. Values are cited and represented as medians.

S. aureus 15981 adherence was 942-fold lower on MAPTMS-TMOS coating than on uncoated titanium.

According with our results, MAPTMS-TMOS sol-gel coating is a promising antiadherent surface for S. aureus. More studies are necessary in order to evaluate this property with other species and strains.

Bone-regenerative and biocompatible materials are indicated for the regeneration of bone lost in periodontology and maxillofacial surgery. Bio-Oss is a natural bone mineral for bone grafting of bovine origin and the most common used in this kind of interventions¹. Sil-Oss is a new synthetic nanostructured monetite-based material that is reabsorbed at the same time that is replaced by new bone tissue ². Bacterial infection is one of the complications related to this kind of material. Streptococcus oralis is the most associated oral infecting pathogen to oral surgery³ and Staphylococcus aureus is the most common infecting pathogen to maxillofacial non-oral interventions⁴. Here we evaluated bacterial adherence of two of the most common infecting bacteria of this kind of biomaterial: S. oralis and S. aureus, on Bio-Oss and Sil-Oss.

S. oralis ATCC 9811 and S. aureus 15981 strains were used. Bacterial adherence was evaluated using the modified previously described protocol of Kinnari et al.⁵ that was adapted to our biomaterial. The quantification was performed by the drop plate method⁶. The statistical data were analyzed by pairwise comparisons using the nonparametric Mann-Whitney test with a level of statistical significance of p<0.05. Values are cited and represented as medians.

Bacterial adherence decreased significantly on Sil-Oss compared to Bio-Oss. S. oralis ATCC 9811 adherence was between 11 and 13-fold less on Sil-Oss compared to Bio-oss. In the case of S. aureus15981, the adherence was between 4 and 6-fold less on Sil-Oss compared to Bio-Oss.

Sil-Oss nanostructured monetite-based biomaterial could be considered as a promising biomaterial to be used for the regeneration of bone defects since the bacterial adherence on it is lower than on another currently used material.

Summary Statement

Description of an original in vitro protocol for assessing combined bacteria and cell competitive adherence on the surface of biomaterials of medical interest

Summary Statement

Combination of antibiotics with N-acetylcisteine and sub-MIC concentration of erythromycin was evaluated in two collection and 16 clinical strains of staphylococci isolated from PJI. The results were strain-dependent, so it evidences the necessity of perform individual studies of biofilm susceptibility.

Summary Statement

A study to evaluate biofilm development on different coatings of UHMWPE was performed. We observed a species-specific effect, with S. aureus affected mainly by DLC-F and S. epidermidis by DLC. These data correlates with previous adherence studies.

Introduction: Papers about high-virulence infections are not usual, results contradictory, and orthopaedic outcomes not described.

Purpose: to compare infectious and orthopaedic results after late arthroplasty infections by single vs. polymicrobial isolates, low vs. high-virulence, and Gram-positive vs. Gram-negative organisms, when treated by exchange surgery plus long cycles of combined oral antibiotics.

Patients and Methods: A late arthroplasty infection was diagnosed in 68 consecutive patients (48 female) of 72.2(+/−10.2) years (37 hips/31 knees).

Cultures were polymicrobial in 22 cases and by Gram-positive in 55 (80.9%). Highly-resistant organisms: methicillin-resistant Staphylococcus (36 patients) and ESBL-producing Enterobacteriaceae (2 patients). “Problematic-treatment”: Enterococcus (6 patients), Pseudomonas (3 patients), non-fermenting Gram-negative (2), moulds (1).

Oral antibiotic selection: according to bacterial sensitivity, biofilm and intracellular effectiveness. Protocolized surgery: two-stage exchange. Average follow-up: 4.7+/−2.7 years (1–11).

Healing of infection is diagnosed if absence of clinical, serological and radiological signs of infection during the whole follow-up. Orthopaedic outcome is evaluated by HHS for hips and by KSCRS for knees.

Results: Surgery was not possible in 7 infections (rejected by patients), and reimplantation in 17 additional cases (patients died shortly after first surgery, rejected 2nd surgery, or was contraindicated because of medical reasons).

Healing of infection: 59/68 patients (86.8%), 32/37 hips (86.5%) and 27/31 knees (87.1%). Infection not healed: 7/68 cases (10.3%) (4/37 hips, 3/31 knees) (5 by highly-resistant and 1 by “problematic-treatment” bacteria). There are no differences between hips and knees (p=0.55).

Orthopaedic Results: HHS averages 80.5+/−16.2 (81+/−16 in healed infection, 56+/−23.5 in persistent infection). KSCRS averages 77.2/58.1 +/− 19.8/24.5 in healed infections, 32.6/0+/−25.8/0 in persistent infections. Infective and orthopaedic results present a strong statistical correlation in hips (p=0.016) and knees (p=0.0001).

Statistically significant differences are not found when comparing subgroups according to Gram stain (p=0.43), multiple vs single bacteria (p=0.47 infective, p=0.71 orthopaedic), highly-resistant bacteria (p=0.2 infective, p=0.1/0.5 orthopaedic), or “problematic-treatment” (p=0.68).

Conclusions:

A strong statistical correlation appears between infective and orthopedic results after late arthroplasty infections.

Introduction: The x-ray test, introduced at the beginning of the XX century, originated a succession of descriptions of alterations in the different secondary ossification nuclei of the long bones, systematically considered as osteocondrosis cases. Osteocondrosis is a wide concept including etiological, pathological, histological, clinical and radiological data, there being no unique criterium about the concept in the literarure. There are no clear data in the literature about the prevalence of radiological alterations in the forefoot ossification nuclei. In most cases such ‘alterations’ are rather anatomical variants in the development and growth of the ossification nuclei.

The aim of the present study was to determine the different radiological alterations observed in our series and their possible relation with other variables (sex, foot pathology and forefoot morphology).

Material and Methods: A serie of 971 dorso-plantar radiographs from 225 patients were retrospectively analysed. The presence, or lack, of each nucleus and its radiological aspect were observed. The different alterations of the nuclei were classified as: normal, sclerotic, sclerotic and notch, sclerotic and flattened, sclerotic and fragmented, and fragmented. Likewise, a statistical analysis was performed relating the alterations of each nucleus with the forefoot morphology (digital and metatarsal formulae) and the main pathologies motivating the x-ray examination (traumatism, our control group; flatfoot; hallux valgus; clubfoot).

Results: We could not find any asymmetry or dimorphism in our series. The prevalence of different alterations of each nucleus was higher in younger children, excepting in the ossification nucleus of the proximal phalanx.

In the 46.3% of the cases there are radiological alterations in the ossification nucleus of the proximal phalanx. In such cases, the 79.2% were sclerotic.

In the flatfoot patients a higher frequency regarding the apperance of radiological alterations was shown significant (p< 0.05) for first cuneiform, proximal metatarsal, and proximal phalanx nuclei.

In the cases with evident alterations of the proximal or distal metatarsal nuclei, the 100% of the cases was related to egyptian digital formula.

The retrospective study did not provide us with additional clinical information about symptoms that could define osteocondrosis in each case.

Conclusions: There is a higher prevalence regarding to radiological alterations of the ossification nuclei of the first radio of the foot.

The biomechanical alterations of the gait in the flat-foot patients, or its treatment (insole), could be related to radiological alterations of such nuclei.

Introduction: The study of appearance and development of the different ossification nuclei of the skeleton in the diverse segments of the locomotor apparatus is relevant for fields of medicine, such as Human Anatomy, Paediatry, Endocrinology, Forensic Medicine, Traumatology and Orthopaedic Surgery, among others.

A number of studies show significant differences regarding their results due to the heterogeneity of methods and scientific and geographic fields originating each series.

The present study is intended to show the age of appearance and complete fusion of the different ossification nuclei of the first radio of the foot in a present Mediterranean sample of children and its relation with several morphometric and clinical parameters.

Material and Methods: We report a retrospective study where 971 x-ray dorso-plantar images from 225 patients were analysed.

A descriptive and qualitative assessment allowed us to determine the existence or lack of each ossification nucleus of the first radio of the foot. Risser’s scale, adapted by us, was applied in the following way: 0, lack; I, rudimentary nucleus; II, well formed nucleus; III, partial fusion to diaphysis; IV, complete fusion.

Likewise, a statistical analysis was performed relating the ages of appearance and fusion of each nucleus with the forefoot morphology (digital and metatarsal formulae) and the main pathologies motivating the x-ray examination (traumatism, our control group; flatfoot; hallux valgus; clubfoot).

Results: Data about the age of appearance and fusion of each nucleus of the first radio of the foot were quantified.

As a general rule, the age of appearance of each nucleus was earlier in girls. A delay in the age of appearance of the proximal metatarsal epiphysis in clubfoot patients (3.33 years) was observed in comparison with the control group (1.96 years).

In general, there was no relation between sex and the fusion (partial and total) of each nucleus of the first radio of the foot. The exception was the age of appearance of the distal metatarsal epiphysis (when this incostant nucleus was present), earlier in boys (9.49 years) than girls (11.21 years).

A delay in the age of fusion of the proximal and distal metatarsal epiphyses and the proximal phalanx epiphysis was observed in hallux valgus patients.

In patients with egyptian foot, there seems to be a delay in the age of fusion of the distal metatarsal and distal phalanx epiphyses.

Conclusions: The different ages of appearance and partial and total fusion of each ossification secondary nucleus of the first radio of the foot are detailed in the present comunication. A delay in the age of fusion of the secondary nucleus of the first radio of the foot would contribute to determine significantly forefoot morphology (egyptian formula) and even its pathology (hallux valgus).

Biofilm development is a major factor in the pathogenesis of implant-related infections. However, there are only a low number of studies that analyses the ability of clinical isolates of bacteria to develop biofilm in vitro. Here we study biofilm development in several strains of Staphylococcus aureus and Coagulase-negative Staphylococcus (CNS) consecutively isolated from retrieved orthopaedic implants from patients diagnosed of implant-related infections.

We have evaluated in vitro biofilm development using the crystal violet technique in microtiter plates. Biofilm development was confirmed by visual microscopy and Confocal Laser Scanning Microscopy. Staphylococcal strains were isolated from implant-related infections by sonication of retrieved prosthesis as previously published by our group, and identified using conventional methods.

Twenty-seven strains (15 S. aureus, nine S. epidermidis, and one each of S. hominis, S. lugdunensis and S. warneri) were included in the study. Four strains of S. aureus (26.7 %) and one strain of S. epidermidis (8.3 %) did not develop biofilm in the test, showing OD lectures almost identical to the negative control. No statistical differences were detected between the two groups. The microscopic examination confirms this finding. Among the biofilm-producing strains, an important difference of the amount of biofilm produced was detected. One strain (S. aureus) produced biofilm in greater amount than all other strains, detectable even by visual examination of the plate.

In conclusion, not all staphylococcal strains isolated from implant-related infections are able to develop biofilm in vitro. There must be other pathogenic factors that are important in the pathogenesis of implant-related infections and need to be studied in order to develop a better strategy for treat these infections.