Antimicrobial Activity of the Most Common Antibiotic-Releasing Systems Employed in Current Orthopedic Surgery: in vitro Study.

PURPOSE OF THE STUDY Infections of joint replacements represent one of the most serious problems in contemporary orthopedics. The joint infections treatment is usually multimodal and involves various combinations of drug delivery and surgical procedures. The aim of this study was to evaluate and compare the bacteriostatic and bactericidal properties of the most common antibiotic carriers used in orthopedic surgery: bone cements mixed with antibiotic and porous calcium sulfate mixed with antibiotic. MATERIAL AND METHODS Three commercial bone cements (Palacos®, Palacos® R+G, Vancogenx®) and commercial porous sulfate (Stimulan®) were prepared with a known concentration of vancomycin (a glycopeptide antibiotic). Specifically, for the purpose of our study, the testing specimens were prepared to release 0, 1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 mg of vancomycin into 1 liter of solution. The specimens with increasing amount of antibiotic were placed in a separate tubes containing 5 mL of Mueller-Hinton broth inoculated with a suspension (0.1 m, McFarland 1) of the reference strain CCM 4223 Staphylococcus aureus to evaluate their bacteriostatic properties (broth dilution method). After this initial incubation and evaluation of the broth dilution method, an inoculum from each tube was transferred onto blood agar plates. After another 24-hour incubation under the same conditions, we evaluated the bactericidal properties (agar plate method). As many as 132 of independent experiments were performed (4 specimens × 11 concentrations × 3 repetitions = 132). RESULTS The bacteriostatic properties of all investigated samples were excellent, perhaps with the exception of the first bone cement (Palacos®). The sample Palacos® started to exhibit bacteriostatic properties at concentrations ≥ 8 mg/mL, while all other samples (Palacos R+G®, Vancogenx®, and Stimulan®) were bacteriostatic in the whole concentration range starting from 1 mg/mL. The bacteriocidic properties did not show such clear trends, but correlated quite well with different properties of the investigated samples during mixing - the most homogeneous samples seemed to exhibit the best and the most reproducible results. DISCUSSION The reliable and reproducible comparison of ATB carriers is a difficult task. The situation is complicated by high numbers of local antibiotic carriers on the market, numerous antibiotics used, and differences in clinical trials at different laboratories. Simple in vitro testing of bacteriostatic and bacteriocidic properties represents a simple and efficient approach to the problem. CONCLUSIONS The study confirmed that the two most common commercial systems used in the orthopedic surgery (bone cements and porous calcium sulfate) prevent bacterial growth (bacteriostatic effect), but they may not be 100% efficient in complete elimination of bacteria (bacteriocidic effect). The scattered results in the case of bacteriocidic tests seemed to be connected with the homogeneity of ATB dispersion in the systems and with the lower reproducibility of the employed agar plate method. Key words: local release of antibiotics; bone cements; calcium sulfate; antimicrobial susceptibility.

Combining branched copolymers with additives generates stable thermoresponsive emulsions with in situ gelation upon exposure to body temperature.

Branched copolymer surfactants (BCS) containing thermoresponsive polymer components, hydrophilic components, and hydrophobic termini allow the formation of emulsions which switch from liquid at room temperature to a gel state upon heating. These materials have great potential as in situ gel-forming dosage forms for administration to external and internal body sites, where the emulsion system also allows effective solubilisation of a range of drugs with different chemistries. These systems have been reported previously, however there are many challenges to translation into pharmaceutical excipients. To transition towards this application, this manuscript describes the evaluation of a range of pharmaceutically-relevant oils in the BCS system as well as evaluation of surfactants and polymeric/oligomeric additives to enhance stability. Key endpoints for this study are macroscopic stability of the emulsions and rheological response to temperature. The effect of an optimal additive (methylcellulose) on the nanoscale processes occurring in the BCS-stabilised emulsions is probed by small-angle neutron scattering (SANS) to better comprehend the system. Overall, the study reports an optimal BCS/methylcellulose system exhibiting sol-gel transition at a physiologically-relevant temperature without macroscopic evidence of instability as an in situ gelling dosage form.

Structuring and de-structuring of nanovectors from algal lipids. Part 1: physico-chemical characterization.

Lipid nanocarriers are among the most employed systems for drug delivery purposes in several research and industrial sectors, since their favorable properties ensure broad applicability. The design and characterization of these nanosystems are of paramount importance to obtain controlled outcome, since the supramolecular structure and molecular interactions deeply impact the functionality of the resulting aggregates. The choice of the most appropriate formulation for the target of interest relies on in-depth physico-chemical characterization in order to optimize stability, loading rates and sustained release. Several supramolecular architectures suited for carrier development can be obtained from lipid building blocks, by varying lipid composition and packing parameter. In particular, cubosome and liposome aggregates are often used as drug vectors thanks to their high cargo capability and biocompatibility. Moreover, the possibility to employ lipids from natural sources i.e. biomasses to prepare nanosystems makes them especially attractive. In this work, two aggregate types were characterized and compared as drug vectors for poorly water-soluble antioxidants, particularly curcumin and two adjuvants (i.e. tocopherol and piperine). The nanovectors were obtained by extracting lipids from algal biomasses with different lipid composition, and characterized by advanced structural (DLS, SAXS, Cryo-TEM) techniques, spectroscopy (NMR) and calorimetry (ITC). Finally, the structural stability of both aggregate types was evaluated.

[Repeated Sterilization of Articular Components of Joint Replacements with Ethylene Oxide Does Not Affect the Structure and Properties of UHMWPE]. / Opakovaná sterilizace artikulacních komponent kloubních náhrad etylenoxidem nemá vliv na strukturu a vlastnosti UHMWPE.

PURPOSE OF THE STUDY In clinical practice UHMWPE is the most commonly used material for manufacturing articular components of joint replacements. The purpose of this study is to find out whether repeated ethylene oxide sterilization results in oxidative degradation of UHMWPE or not and also whether the oxidative degradation of various types of ethylene oxide-sterilized UHMWPE depends on storage time or not. MATERIAL AND METHODS The set included 12 samples of UHMWPE (three samples with different modifications (virgin PE, with E vitamin and cross-linked with thermal treatment) and different number of sterilizations (0×-3×)). The set also included 8 samples of commercial components of hip or knee replacements sterilized with ethylene oxide and stored for different storage periods. The oxidative degradation was assessed by infrared microspectroscopy, based on which the oxidation index (OI), transvinylene index (VI), crystallinity index (CI) and E vitamin index (EI) were calculated. Mechanical properties of UHMWPE were obtained through microhardness measurements. Statistical processing of the results was performed. RESULTS In all the samples, very low oxidative degradation values were reported (most OI values < 0.1). All radiation crosslinked UHMWPE samples showed an increased VI index and a slightly lower crystallinity index. All unmodified samples (irrespective of whether or not and how many times or how long ago the samples were sterilized with EtO) had almost zero value of VI. Changes in crystallinity were negligible (in the rage of 0.56-0.63), which required very accurate measurements of micromechanical properties. Yet, linear correlation was established between microhardness and crystallinity. DISCUSSION All the mentioned indices changed as anticipated: OIs were very low and slightly increased with time of storage, VIs of radiation crosslinked samples grew in proportion to the total gama radiation dose, CIs decreased in samples thermally treated by remelting, and EIs were very low due to negligible concentration of stabiliser (0.1%) in the samples of medical grade UHMWPE. CONCLUSIONS All samples showed zero or minimum oxidative degradation. This confirmed that neither ethylene oxide sterilization, nor multiple EtO sterilization or longer storage of polymer after ethylene oxide sterilization result in major oxidative degradation. Key words: UHMWPE, ethylene oxide, sterilization, oxidation, infrared spectroscopy, microhardness.

Polyethylenimine based magnetic nanoparticles mediated non-viral CRISPR/Cas9 system for genome editing.

Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology- directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.

[Comparison of the Quality of the Most Frequently Used New UHMWPE Articulation Inserts of the Total Hip Replacement]. / Porovnání kvality nejcasteji pouzívaných nových UHMWPE artikulacních vlozek náhrady kycelního kloubu.

PURPOSE OF THE STUDY For an orthopedic surgeon it is difficult or even impossible to estimate the real quality of ultrahigh molecular weight polyethylene (UHMWPE) liners that are used in the total joint replacements (TJR) just on the basis of information given by the manufacturers. At the same time, the quality of the UHMWPE liner can impact strongly on the total lifespan of the implanted TJR. This work aims at independent, objective comparison of properties of the UHMWPE liners for total hip replacements (THR), which are most frequently used in the Czech Republic. MATERIAL AND METHODS We analyzed seventeen most frequently implanted UHMWPE cups of different manufacturers implanted in the Czech Republic between 2014 and 2015 and four control samples prepared by standard industrial-scale procedures according to our instructions, whose modification (crosslinking, thermal treatment, stabilization and sterilization) was known in detail. The UHMWPE polymer was characterized by four independent microscale methods, suitable for relatively small and irregular specimens such as THR cups: infrared microspectroscopy (IR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). RESULTS The employed methods yielded quite complete information about the investigated UHMWPE materials. IR spectra enabled us to calculate oxidation indexes (OI, measure of oxidative damage), trans-vinylene indexes (VI, measure of absorbed radiation dose during crosslinking and/or sterilization) and crystallinity indexes (CI, amount of crystalline phase that strongly influences mechanical performance). DSC curves were employed in calculation of crystallinities (wc, proportional to CI) and melting points (Tm, proportional to the average thickness of crystalline lamellae). MH measurements confirmed that the observed structure changes showed a real impact on mechanical properties. TGA experiments gave rough estimate of stabilization and, consequently, possible long-term oxidation resistance. Significant correlations among oxidative damage (OI), crystallinity (CI, wc) and microhardness (Hv) were statistically proven. The highest oxidative degradation was usually observed in samples thermally treated by annealing and/or sterilized by gamma irradiation. DISCUSSION The results confirmed our expectations that the UHMWPE liners from various manufacturers can be significantly different as far as their molecular structure, supermolecular structure, and mechanical properties are concerned. The differences among the various UHMWPE can be expected to increase after the implantation during in vivo. CONCLUSIONS From the clinical practice point of view, the results showed the following facts: (i) In the field of THR, all manufactures prefer crosslinked types of UHMWPE due to their increased wear resistance; non-crosslinked UHMWPEs are regarded as obsolete. (ii) Most of the manufacturers prefer ethylene oxide or gas plasma sterilization to gamma sterilization because the gammasterilized UHMWPEs exhibit lower long-term oxidation resistance. (iii) Modern trend is the stabilization of UHMWPEs with vitamin E. Key words: UHMWPE, hip replacements, oxidative degradation, infrared spectroscopy, microhardness.

[Comparison of Explanted UHMWPE Hip Replacement Components of Various Manufacturers after 10 Years in vivo]. / Porovnání explantovaných UHMWPE komponent náhrady kycelního kloubu ruzných výrobcu po 10 letech in vivo.

PURPOSE OF THE STUDY Considered to be one of the most common causes of aseptic loosening of endoprostheses is the THA failure due to the wear of articulating components, UHMWPE in particular. The purpose of this study is to verify, in terms of oxidative damage and other parameters, the differences between the UHMWPE implants made by various manufacturers explanted for aseptic loosening with the same life span in vivo. MATERIAL AND METHODS In the period 2010-2015, a total of 21 THA articulating components (cups) made of Ultrahigh molecular weight polyethylene by seven different manufacturers were explanted. For each manufacturer, three UHMWPE cups with the same life span (10-12 years after the primary implantation) were evaluated. The damage to the examined joint replacements was described in complexity using three different criteria, namely independently by three evaluators - experienced orthopaedic surgeons. The evaluated criteria were the following: degree of osteolysis determined based on the preoperative radiographs, wear rate of the explanted UHMWPE component, and extent of perioperatively detected granuloma. Oxidative damage and other structural characteristics of explanted cups were studied by means of infrared spectroscopy and microhardness testing. The correlation between the clinical orthopaedic assessment and oxidative damage were statistically processed. RESULTS Strong correlations between the oxidative damage and crystallinity, strong correlations between all types of orthopaedic assessments, negligible correlations between trans-vinylene index and all the other quantities, and moderate correlations between the oxidative damage and clinical evaluation were identified. It was confirmed by experimental measurement that the observed high oxidative damage, resulting in increased crystallinity, manifested itself also in micromechanical properties of the material at the respective site of the THA articulating component. DISCUSSION The discussion includes the comparison of correlations of individual quantities as well as potential effects on the differences in values of components made by individual manufacturers. The values are related to the data in literature and generally accepted claims. CONCLUSIONS At the time of failure almost all the components showed severe or even critical oxidative damage that strongly correlated with the overall clinical evaluation of the damage to the implant. This confirmed that the oxidative degradation is one of the main causes of THA failure. Key words: UHMWPE, oxidation index, crystallinity, THA failure, wear.

[The Risk of Total Joint Replacement Infectious Complications in Relation to the UHMWPE Particle Adhesion Area]. / Riziko infekcních komplikací po náhrade nosného kloubu ve vztahu k adhezní plose UHMWPE cástic.

PURPOSE OF THE STUDY Periprosthetic joint infection is a major complication which in most of the cases requires a long-term administration of antibiotics and often necessitates undergoing multiple challenging surgeries. Bacterial adhesion to foreign material is one of the key risk factors associated with periprosthetic joint infection. The foreign material with large adhesion area might be also the UHMWPE (Ultrahigh molecular weight polyethylene) particles released during the wear process from the surface of articulating components. The purpose of this study is to evaluate potential adhesion areas of wear particles in relation to diverse distribution of the size and shapes of wear particles in periprosthetic tissue and to assess an increase in the risk of infectious complications associated with an increase in the adhesion area of wear particles. MATERIAL AND METHODS The size and morphology of model and real UHMWPE particles were determined with the use of light microscopy and scanning electron microscopy. By determining the morphological descriptors, the surfaces of individual particles for different distributions of polyethylene particles were calculated. When measuring the model wear particles, 6 model situations were simulated, in which comparisons with the control measurement by the BET (Brunauer-Emmet-Teller) method were made. RESULTS The variability of individual morphological descriptors demonstrates the effect on the total surface of particles. The calculated coefficient defines how many times the particle surface increases when corrected to the given descriptor (elongation, flattening, roughness, porosity). The total area of real wear particles at 1 year is 4,622 cm2, at 20 years it is 92,440 cm2. Based on our calculations, the area of particles where a biofilm is actually formed (approximately 50 bacteria may adhere to a particle of 3µm in diameter) is 809.5 cm2 at 1 year and 16,190 cm2 at 20 years. DISCUSSION According to the measurements, the size of the potential adhesion area of metal parts and polyethylene particles becomes equal already after several weeks of endoprosthesis usage and after a few years it is many times larger. The question is whether the risk of bacterial adhesion, i.e. also the risk of infectious complications of TEP actually increases. The clinical practice suggests that the number of infections e.g. 10, 15 or 20 years after the primary implantation is not statistically higher, despite the confirmed growth of potential adhesion area in the form of UHMWPE particles. This fact could be explained by a partially equal regulatory pathway of infection and polyethylene disease. The immune system stimulated by wear particles might better resist the hematogenic infection. CONCLUSIONS The study outcomes clearly indicate that the area of polyethylene wear particles considerably increases over time. In spite of the fact that only approximately 10% of wear particles show parameters (also with respect to the size of particles and bacteria) for potential bacterial adhesion, this area is many times larger than the area of metal parts of the endoprosthesis. Key words: UHMWPE particle, adhesion, biofilm, wear, TJR infection.

Thermoplastic starch composites with TiO2 particles: Preparation, morphology, rheology and mechanical properties.

Composites of thermoplastic starch (TPS) with titanium dioxide particles (mTiO2; average size 0.1µm) with very homogeneous matrix and well-dispersed filler were prepared by a two-step method, including solution casting (SC) followed by melt mixing (MM). Light and scanning electron microscopy confirmed that only the two-step procedure (SC+MM) resulted in ideally homogeneous TPS/mTiO2 systems. The composites prepared by single-step MM contained non-plasticized starch granules and the composites prepared by single-step SC suffered from mTiO2 agglomeration. Dynamic mechanical measurements showed an increase modulus with increasing filler concentration. In TPS containing 3wt.% of mTiO2 the stiffness was enhanced by >40%. Further experiments revealed that the recommended addition of chitosan or the exchange of mTiO2 for anisometric titanate nanotubes with high aspect ratio did not improve the properties of the composites.

[Analysis of Oxidative Damage to Components Removed from Beznoska/Poldi Total Hip Replacements]. / Analýza oxidacního poskození explantovaných komponent náhrady kycelního kloubu Beznoska/Poldi.

UNLABELLED: PURPOSE OF THE STUDY Ultrahigh molecular weight polyethylene (UHMWPE) is today the most frequently used bearing surface in total joint replacements (TJR) because of its properties, i.e., excellent biocompatibility, good mechanical and tribological performance and high wear resistance. UHMWPE liners are the most loaded TJR components and, therefore, their properties are decisive for TJR longevity. This study had three objectives: 1) to evaluate the oxidative degradation of explanted UHMWPE components; 2) to look for a statistically significant relationship between the extent of oxidative degradation and the durability of joint replacements; and 3) to investigate whether the durability of a TJR was related to the type of sterilisation used in manufacture. MATERIAL AND METHODS The study included 26 acetabular components obtained at revision arthroplasty between 2004 and 2013 from patients in whom a Beznoska/Poldi total hip replacement was used in the period from 1977 to 2002. The average age of the patients at the time of primary implantation was 57.9 years, the average longevity of the components removed was 18.63 years (range, 6.9 to 27.9 years). Samples of worn out and unworn areas from explanted components were processed in a three-step procedure in order to finally obtain 2-mm microtome sections. These were studied by infrared microspectroscopy. Oxidative damage to UHMWPE was determined as the oxidation index (OI); radiation damage to UHMWPE during sterilization was evaluated as the transvinylene index (VI); oxidation-induced changes in the polymer structure and its properties were characterised as the crystallinity index (CI); and local changes in mechanical properties due to oxidative degradation were assessed as microhardness (MH). Spearman's correlation coefficient and the Wilcoxon two-sample test were used for statistical analysis. RESULTS The OI values (average and maximum) in both worn out and unworn surface areas were related to component longevity. The difference between the oxidation index of ruptured components and that of the other components was statistically significant. Significant differences were also found between the average and maximum OI values of worn out areas and those of unworn surfaces. The relationship between the average oxidation index in both the unworn and worn out areas of UHMWPE components and the longevity of cracked components was statistically significant. DISCUSSION Our results show that the OI values obtained by microspectroscopy correlated with both the microscopic damage and the longevity of UHMWPE liners and the correlation was statistically significant also in relation to the longevity of total replacements. Relationships amongst OI, VI, CI and MH values as well as their relation to failure and longevity of total replacements are discussed. CONCLUSIONS It can be concluded that infrared microspectroscopic measurement of OI values is a simple and fast method to characterise UHMWPE liners. In addition, the IR spectra also show other supplementary characteristics, such as VI and CI indices. These values provide information on the quality of various UHMWPE types currently used in TJR surgery. The types of UHMWPE which exhibit high oxidative degradation should be avoided in clinical practice due to increased risk of early TJR failure. Responsible orthopedic surgeons should be aware of this fact and, if possible, collaborate with an independent, noncommercial laboratory in order to evaluate the quality of various UHMWPE liners used in their hospitals. KEY WORDS: UHMWPE, oxidation, total joint replacement, infrared spectroscopy, microhardness.

Silica-modified monodisperse hexagonal lanthanide nanocrystals: synthesis and biological properties.

Oleic acid-stabilized hexagonal NaYF4:Yb(3+)/Er(3+) nanocrystals, emitting green and red luminescence, were prepared by the high-temperature co-precipitation of lanthanide chlorides. By varying the reaction time and the Ln(3+)/Na(+) ratio, the nanocrystal size can be controlled within the range 16-270 nm. The maximum upconversion quantum yield is achieved under 970 nm excitation. The reverse microemulsion technique using hydrolysis and condensation of tetraethoxysilane is a suitable method to coat the nanocrystal surface with a silica shell to make the particles dispersible and colloidally stable in aqueous media. During the subsequent functionalization, (3-aminopropyl)trimethoxysilane introduced amino groups onto the silica to enable future bioconjugation with the target molecules. All specimens were characterized by TEM microscopy, electron and X-ray diffraction, ATR FT-IR spectroscopy, and upconversion luminescence. Finally, in vitro cytotoxicity and intracellular nanoparticle uptake (using confocal microscopy) were determined with human cervical carcinoma HeLa and mRoGFP HeLa cells, respectively. From the investigated particles, amino-functionalized NaYF4:Yb(3+)/Er(3+) nanocrystals internalized into the cells most efficiently. The nanoparticles proved to be nontoxic at moderate concentrations, which is important when considering their prospective application in biolabeling and luminescence imaging of various cell types.

[Effect of sterilisation with formaldehyde, gamma irradiation and ethylene oxide on the properties of polyethylene joint replacement components]. / Vliv sterilizace formaldehydem, gama zárením a etylenoxidem na vlastnosti polyetylenových komponent kloubních náhrad.

PURPOSE OF THE STUDY: Each method of sterilisation has some effect on the structure and properties of UHMWPE and thus also on joint replacement longevity. This study was designed to compare, using objective methods of measurement, several kinds of sterilisation and to recommend the one which has the best prospect for making joint replacements last longer. MATERIAL AND METHODS: Two groups of UHMWPE samples were tested. Group 1 included virgin GUR 1020 polyethylene, non-modified and non-sterilised (Meditech, Germany). Group 2 comprised of three sets of samples sterilised with formaldehyde, gamma irradiation and ethylene oxide, respectively. In both groups, physicochemical properties were assessed by infrared spectroscopy (IR), and the oxidation (OI) and trans-vinyl (VI) indices, which show the degree of oxidation of a material, were determined. Free-radical concentrations were measured by the method of electron spin resonance (ESR). The mechanical properties of each sample were studied using small punch tests (SPT) and testing microhardness (MH). Any change in mechanical properties can affect, to various degrees, the quality and longevity of a prosthetic joint. RESULTS: The samples sterilised by gamma irradiation showed higher values of both the OI (0.37) and the VI index (0.038) than the other samples (OI, 0.02 to 0.05 and VI, 0). Also, the free-radical concentration was detectable only in the gamma-sterilised sample. Values obtained for mechanical properties were as follows: peak load in the range of 58.48 N (gamma irradiation) to 59.60 N (ethylene oxide); ultimate load in the range of 46.69 N (gamma irradiation) to 57.50 N (ethylene oxide); ultimate displacement in the range of 4.29 mm (gamma irradiation) to 4.58 mm (virgin polyethylene and formaldehyde); and work to failure in the range of 185.18 mJ (gamma irradiation) to 205.89 mJ (virgin polyethylene). Microhardness values were obtained in the following ranges: 41.2 to 44.6 MPa (virgin polyethylene); 40.2 to 44.1 MPa (formaldehyde); 46.1 to 49.3 MPa (gamma irradiation); and 40.3 to 44.2 MPa (ethylene oxide). DISCUSSION: The samples sterilised with formaldehyde and ethylene oxide have mechanical properties very similar to virgin polyethylene, they are not damaged by oxidation and do not contain free radicals. Owing to these characteristics, the immediate and long-term oxidation stability of the three samples is higher. The sample sterilised by gamma irradiation showed the presence of free radicals and immediate and long-term oxidative degradation. This results in the deterioration of mechanical properties and the growth of crystallinity due to enhanced oxidation and leads to higher polyethylene microhardness. CONCLUSIONS: Sterilisation with gamma irradiation results in oxidative degradation and mechanical property deterioration, which is one of the potential risks of a shorter life span of joint replacements. The use of ethylene oxide or formaldehyde does not change polymer properties nor has any effect on oxidation of materials. Therefore, a longer life expectancy of the joint replacements sterilised with ethylene oxide can be expected. The life span of their joint replacements is a key issue for the patients. Prosthetic joint loosening is painful and the patient often requires re-implantation. A higher number of re-implantations is associated with higher costs for the institution involved and, consequently, for the whole health care system. Although this study basically deals with chemical issues, it informs the surgeon of the latest developments leading to the improvement of implanted materials, which can increase the life expectancy of joint replacements and patients' satisfaction.

Simultaneous detection of multiple targets for ultrastructural immunocytochemistry.

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.

[Current knowledge on the effect of technology and sterilization on the structure, properties and longevity of UHMWPE in total joint replacement]. / Soucasné poznatky o vlivu technologie výroby a sterilizace na strukturu, vlastnosti a zivotnost UHMWPE v kloubních náhradách

Ultrahigh molecular weight polyethylene (UHMWPE) is the most frequently used bearing surface in currently used total joint replacements (TJR). According to the literature available, UHMWPE is the best polymer material, in terms of biocompatibility, mechanical properties and wear resistance, for this application. In spite of this fact, UHMWPE wear (i.e., release of microscopic particles from the polymer surface) remains one of the main reasons of TJR failures. Consequently, the wear of UHMWPE is a subject of intensive study by both materials scientists and orthopaedic surgeons. The structure and properties of UHMWPE strongly depend on the way of processing and post-processing modifications. The processing includes polymer resin preparation (microparticles about 100 em in size) and resin consolidation (forming bulk material). Post-processing modifications aim at increasing wear resistance and oxidation stability which are regarded as major factors involved in TJR failure. In order to maintain high purity materials for medical application, it is not allowed to use additional chemicals during the modification processes. The only exception is the use of vitamin E, a natural stabilizer and antioxidant. Considering all the above mentioned facts, the modifications can be based on (I) ionizing radiation such as gamma rays or accelerated electrons, (II) thermal modification, (III) additional stabilization with vitamin E, and (IV) sterilization. According to the modifications, we usually differentiate three generations of UHMWPE. The 1st generation UHMWPE is not modified except for obligatory sterilization. The sterilization procedures based on chemical procedures (formaldehyde vapours, hot water) have lately been forbidden, abandoned and replaced by gamma-irradiation with doses of 25-45 kGy. In the course of time, sterilization by means of gamma-irradiation showed to be unsuitable due to oxidative degradation of UHMWPE, which resulted in lower wear resistance, worse material properties and a decrease in longevity of joint replacements. The 2nd generation UHMWPE or highly cross-linked UHMWPE is material treated with ionizing radiation (gamma-rays or accelerated electrons) at higher doses (usually 50-100 kGy). The irradiation is followed by thermal treatment in order to eliminate residual radicals and limit oxidative degradation that occurs in the 1st generation UHMWPE types. Finally, the material is sterilized usually by modern procedures using ethylene oxide or gas plasma neither of which causes oxidative degradation (as opposed to gamma-irradiation sterilization). The 3rd generation UHMWPE is basically the same as that of the 2nd generation. The main difference consists in slightly better mechanical properties (strength, fatigue) and further stabilization to oxidative degradation. This is achieved by special procedures, some of which are either controversial (sequential irradiation) or well-accepted by a great majority of the orthopaedic community (vitamin E stabilization). Only the future will show whether the difference between the 2nd and 3rd generations of UHMWPE will play any role in increasing TJR longevity and therefore the quality of its performance. The final objective of all of UHMWPE modifications is, as already mentioned, to increase wear resistance, oxidative resistance and mechanical performance of the polymer. These changes, especially an increase in wear resistance, have been confirmed to prolong the longevity of total joint replacements based on UHMWPE.

Simple colorimetric methods for determination of sub-milligram amounts of ultra-high molecular weight polyethylene wear particles.

New colorimetric methods are described for determination of sub-milligram amounts of ultra-high molecular weight polyethylene (UHMWPE) wear particles. These methods are based on the irreversible binding of the fluorescein-conjugated bovine serum albumin or the hydrophobic dye Oil Red O to wear particles. UHMWPE particles bind both substances from their solutions and thus decrease the absorbance of these solutions. The decrease is linearly dependent on the amount of added wear particles in the sub-milligram range suitable for practical use. The newly developed method offers improved accuracy and precision compared to Fourier transformed infrared spectroscopy (Slouf M, et al. Quantification of UHMWPE wear in periprosthetic tissues of hip arthoplasty: description of a new method based on IR and comparison with radiographic appearance. Wear 2008;265:674-684.).

[MORF method for assessment of the size and shape of UHMWPE wear microparticles and nanoparticles in periprosthetic tissues]. / Metoda MORF pro sledování velikostí a tvaru oterových mikro- a nanocástic UHMWPE v periprotetických tkáních.

PURPOSE OF STUDY: Aseptic loosening of total joint replacement (TJR) due to wear of ultra-high molecular weight polyethylene (UHMWPE) is regarded as one of the major problems in the field of arthroplasty. This work describes a newly developed method, called MORF, which completely describes the morphology of UHMWPE wear particles. The differences in wear particle morphology may help to elucidate individual differences in TJR failures. MATERIAL AND METHODS: During the years 2002-2010, a set of 47 typical damaged periprosthetic tissues, coming from 16 TJR revisions, was collected. Isolated on polycarbonate (PC) filters were quantified. Quantification of the particles consisted in determination of their concentration and description of their morphology by means of the newly developed MORF method. Firstly, the micrographs of isolated UHMWPE particles were obtained with a scanning electron microscope (Quanta 200 FEG; FEI) at two magnifications: x1200 and x6000. Secondly, both high- and low-magnification micrographs were processed by a standard image analysis software (program NIS Elements; Laboratory Imaging) in order to obtain basic morphological descriptors. Finally, the results from image analysis of high- and low- magnification micrographs were combined by means of our own program MDISTR in order to obtain correct particle sizes and shapes. RESULTS: In the first stage, the method was applied to 25 samples and yielded an average particle size of 0.51 µm. In the second stage, the method was further improved in order to calculate not only the size of particles but also the shape of descriptors. The improved method was applied to eight samples and gave an average size of particles (equivalent diameter, D) in the range of 0.27 - 0.60 µm, circularity (C) of 0.66-0.85 and elongation (E) of 1.75-1.79, suggesting that the great majority of particles were approximately spherical. Finally, in the third stage, the MORF method was applied to two exceptional samples which contained extremely small wear particles (D = 18.5 nm and 21.2 nm). The shape of these small wear nano- particles (C = 0.97 and 0.93; E = 1.29 and 1.35) was even more spherical than that of wear microparticles described abo- ve. This was one of the first two studies which proved the presence of UHMWPE wear nanoparticles in vivo. DISCUSSION: Our newly developed MORF method described in this contribution yields both size and shape descriptors of UHMWPE wear particles, with sizes from 0.1 to 10 µm, which are regarded as most biologically active. The main objective of the method is to yield the highest accuracy. This is achieved by parallel analyses of high- and low-magnification micrographs taken of the same sample. In the end, the two analyses are combined together in order to obtain the correct and complete size and shape description of all particles in the sample. The morphology of UHMWPE wear particles influences TJR lifetime both directly (size and shape of the particles is related to their biological activity) and indirectly (for the given total volumetric wear, size and shape of the particles influence their concentration, which is associated with the biological respon- se of the organism). CONCLUSION: The authors have developed a new method which yields a complete description of the size and shape of UHMWPE wear particles in periprosthetic tissues. The method, which was called MORF, can be applied to studies of TJR failures and also used to evaluate the quality of different UHMWPE components of TJR. The method is quite universal and therefore can be used not only for analyses of wear particles, but also for other types of particles, such as microparticles in polymer blends or inorganic/metallic nanoparticles.

[Comparison of in vivo characteristics of polyethylene wear particles produced by a metal and a ceramic femoral component in total knee replacement]. / Srovnání oterových charakteristik polyetylenu in vivo u náhrad kolena s kovovou a keramickou femorální komponentou.

PURPOSE OF THE STUDY: The aim of the study was to evaluate in vivo and compare, in terms of the quality and number of ultra high-molecular polyethylene (UHMWPE) wear particles, total knee replacements of identical construction differing only in the material used for femoral component production, i.e., CoCrMo alloy or ZrO2 ceramics. MATERIAL AND METHODS: Samples of peri-prosthetic granuloma tissue were collected in two patients with total knee replacement suffering from implant migration, who were matched in relevant characteristics. The primary knee replacement in Patient 1 with a CoCrMo femoral component was done 7.2 years and in Patient 2 with a ZrO2 implant 6.8 years before this assessment. The polyethylene wear-induced granuloma was analysed by the MORF method enabling us to assess the shape and size of wear debris and the IRc method for assessment of particle concentration. RESULTS: In the granuloma tissue samples of Patient 1, on the average, particles were 0.30 mm in size and their relative volume was 0.19. In the Patient 2 tissue samples, the average size of particles was 0.33 mm and their relative volume was 0.26. There was no significant difference in either particle morphology or their concentration in the granuloma tissue between the two patients. DISCUSSION: One of the options of how to reduce the production of polyethylene wear particles is to improve the tribological properties of contacting surfaces in total knee replacement by substituting a cobalt-chrome femoral component with a zirconia ceramic femoral component. The previous in vitro testing carried out with a mechanical simulator under conditions approaching real weight-bearing in the human body did show a nearly three-fold decrease in the number of UHMWPE wear particles in zirconia components. The evaluation of granuloma tissue induced by the activity of a real prosthetic joint for nearly seven years, however, did not reveal any great difference in either quality or quantity of polyethylene debris between the two replacements. The difference of surface roughness between CoCrMo (Ra = 0.05) and ZrO2 (Ra = 0.02) components did not play any role in in vivo conditions. CONCLUSIONS In accordance with a previous clinical study, this evaluation of the quality and quantity of UHMWPE wear particles produced by a ceramic femoral component in vivo failed to demonstrate any advantage of zirconia ceramic components over the cobalt-chrome femoral components so far used.

[Distribution of UHMWPE wear particles in periprosthetic tissues of total hip replacements]. / Distribuce oterových cástic UHMWPE v periprotetických tkáních u TEP kycelního kloubu.

INTRODUCTION: Information on the distribution of UHMWPE wear particles around joint replacements is essential for the understanding of aseptic loosening of implants. MATERIAL AND METHODS: Samples of soft tissue from around total hip arthroplasty (THA), which had a typical appearance of polyethylene granuloma, were collected from the places corresponding to the radiological zones of Gruen and DeLee. The samples were taken from 45 patients (group N1). Since the samples varied in macroscopic appearance and/or the technique of processing, a sub-group of 15 more uniform samples (group N2) were collected by the first author in order to ensure the maxi- mum reliability. The samples were examined for the extent of tissue damage and the amount of particles in each zone. The comprehensive orthopaedic evaluation of tissue damage was carried out as follows: the first author studied the joint replacement revision protocol and pre-operative radiograph in each patient. He evaluated the degree of damage in each zone and wrote down the results in the form of an ordered series according to increasing damage (relational statement). This ordered series was called orthopaedist's statement (OS). The amount of particles in each zone around THA was assessed by the IRc method developed in the laboratory of the second author. IRc method yielded numbers that were proportional to the volume of biologically active particles with size 0.1-10 microm in given zones. The set of numbers from IRc method for each individual case of THA was ordered in a series (relational statement). This ordered series was called a measurement statement (MS) and could be directly compared with orthopaedist's statement (OS). The OS's and MS's for each patient were statistically evaluated, independently for each group (N1=45, N2=15), and two hypotheses were tested: a) distribution of particles in various zones around THA is uniform, b) there is an agreement between OS and MS. RESULTS: The number of UHMWPE wear particles in different periprosthetic zones within each patient was significantly different in both groups (N1 and N2) at the significance level of P=0.001. The OS's and MS's were identical at the significance level P=0.05. Moreover, maximum amount of particles was found in zones III and 7 in 40 patients out of 45. DISCUSSION: Polyethylene wear debris is considered one of the major causes of THR failures. However, neither national nor international literature has provided a study that would show a statistically significant relationship between the numbers of wear particles in individual zones and the extent of tissue damage in these zones. This is most probably due to the fact that four independent methods had to be developed in order to compare OS's and MS's: 1) accurate technique of tissue sampling during THA revisions, 2) accurate method of isolating wear particles from the tissues collected, 3) efficient, reliable and reproducible quantification of UHMWPE particles and 4) correct method of statistical analysis for comparison of the orthopaedist's statements and the measurement statements for all patients involved. CONCLUSION: The distribution of wear particles in the periprosthetic tissues of THA is not uniform, i.e. the amount of wear particles of each patient varies from zone to zone. The extent of tissue damage in each zone, as assessed by the orthopaedic surgeon (OS), was in a good agreement with the volume of 0.1-10 microm particles obtained by IRc measurement (MS) of the zone. We have provided conclusive evidence that the highest number of particles is accumulated in zone III and zone 7. As the number of particles the zones varies and the greatest damage is repeatedly found in areas with the highest particle numbers, it can be concluded with certainly that wear particles are one of the main causes of aseptic joint implant failure.

Distribution of polyethylene wear particles and bone fragments in periprosthetic tissue around total hip joint replacements.

Ultra-high molecular weight polyethylene (UHMWPE) wear particles play a significant role in failures of total joint replacements (TJRs). In this work, we investigated the distribution of these wear particles in periprosthetic tissues obtained from nine revisions of hip TJR. In the first step, all periprosthetic tissues were combined and mechanically separated into granuloma tissue (containing hard granules visible to the naked eye) and surrounding tissue (without visible granules). In the second step, the tissues were hydrolyzed by protease from Streptomyces griseus and granules were separated by filtration; this divided the sample into four groups: (i) lyzate and (ii) non-degraded large granules from the granuloma tissue plus (iii) lyzate and (iv) non-degraded small granules from the surrounding tissue. In the third step, the large as well as small granules were hydrolyzed by collagenase from Clostridium histolyticum. In the last step, the UHMWPE wear particles from all four groups were purified by HNO3 digestion and weighed. The purity of the isolated particles was verified by scanning electron microscopy, infrared spectroscopy and energy-dispersive X-ray analysis. Of the total amount of polyethylene particles in the whole granuloma tissue, 72% of particles in the size range 0.1-10 microm and 68% of those larger than 10 microm were found in granules. Therefore, the formation of granules significantly lowers the effective amount of wear particles available for interaction with reactive cells and seems to be a natural defense mechanism.

[Polyetheretherketone (PEEK). Part I: prospects for use in orthopaedics and traumatology]. / Polyetheretherketon (PEEK) - I. cást: Perspektivní materiál pro ortopedickou a traumatologickou praxi.

On the basis of current studies, polyaryletherketone (PAEK) polymers appear to be materials with great prospects for medical application. The most important member of this family of semi-crystalline thermoplastics is polyetheretherketone (PEEK) with its composites. The excellent properties of this material find their use in the treatment of various disorders of the skeletal system. At present they are used with advantage to construct spinal implants, and replacements for other orthopaedic applications and for dental and trauma medicine are at advanced stages of clinical testing.