Upcycling of Chitin to Cross-Coupling Catalysts: Tailored Supports and Opportunities in Mechanochemistry.

In this study chitin derived from shrimp shells was used in the design of heterogeneous Pd-based catalysts for Heck and Suzuki-Miyaura cross-coupling reactions. The synthesis of Pd nanoparticles supported on N-doped carbons was performed through different approaches, including a sustainable mechanochemical approach, by using a twin-screw extruder. All catalytic systems were characterized by a multitechnique approach and the effect of nanoparticles size, N-doping on the support, and their synergistic interactions were elucidated. Specifically, Kelvin Probe Atomic Force Microscopy provided valuable insights on charge transfer and metal-support interactions. The catalytic behaviour of the samples was investigated in cross-coupling reactions under batch conditions and under semi-continuous flow solvent-free conditions, respectively obtaining a quantitative yield and a noteworthy productivity of 8.7 mol/(gPdh).

Structural heterogeneity of the ion and lipid channel TMEM16F.

Transmembrane protein 16 F (TMEM16F) is a Ca2+-activated homodimer which functions as an ion channel and a phospholipid scramblase. Despite the availability of several TMEM16F cryogenic electron microscopy (cryo-EM) structures, the mechanism of activation and substrate translocation remains controversial, possibly due to restrictions in the accessible protein conformational space. In this study, we use atomic force microscopy under physiological conditions to reveal a range of structurally and mechanically diverse TMEM16F assemblies, characterized by variable inter-subunit dimerization interfaces and protomer orientations, which have escaped prior cryo-EM studies. Furthermore, we find that Ca2+-induced activation is associated to stepwise changes in the pore region that affect the mechanical properties of transmembrane helices TM3, TM4 and TM6. Our direct observation of membrane remodelling in response to Ca2+ binding along with additional electrophysiological analysis, relate this structural multiplicity of TMEM16F to lipid and ion permeation processes. These results thus demonstrate how conformational heterogeneity of TMEM16F directly contributes to its diverse physiological functions.

Imaging single CaMKII holoenzymes at work by high-speed atomic force microscopy.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a pivotal role in synaptic plasticity. It is a dodecameric serine/threonine kinase that has been highly conserved across metazoans for over a million years. Despite the extensive knowledge of the mechanisms underlying CaMKII activation, its behavior at the molecular level has remained unobserved. In this study, we used high-speed atomic force microscopy to visualize the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII with nanometer resolution. Our imaging results revealed that the dynamic behavior is dependent on CaM binding and subsequent pT286 phosphorylation. Among the species studies, only rat CaMKIIα with pT286/pT305/pT306 exhibited kinase domain oligomerization. Furthermore, we revealed that the sensitivity of CaMKII to PP2A in the three species differs, with rat, C. elegans, and hydra being less dephosphorylated in that order. The evolutionarily acquired features of mammalian CaMKIIα-specific structural arrangement and phosphatase tolerance may differentiate neuronal function between mammals and other species.

Antithetic effects of agonists and antagonists on the structural fluctuations of TRPV1 channel.

Transient receptor potential vanilloid member 1 (TRPV1) is a heat and capsaicin receptor that allows cations to permeate and cause pain. As the molecular basis for temperature sensing, the heat capacity (ΔCp) model [D. E. Clapham, C. Miller, Proc. Natl. Acad. Sci. U.S.A. 108, 19492-19497 (2011).] has been proposed and experimentally supported. Theoretically, heat capacity is proportional to a variance in enthalpy, presumably related to structural fluctuation; however, the fluctuation of TRPV1 has not been directly visualized. In this study, we directly visualized single-molecule structural fluctuations of the TRPV1 channels in a lipid bilayer with the ligands resiniferatoxin (agonist, 1,000 times hotter than capsaicin) and capsazepine (antagonist) by high-speed atomic force microscopy. We observed the structural fluctuations of TRPV1 in an apo state and found that RTX binding enhances structural fluctuations, while CPZ binding suppresses fluctuations. These ligand-dependent differences in structural fluctuation would play a key role in the gating of TRPV1.

Light Conversion upon Photoexcitation of NaBiF4:Yb3+/Ho3+/Ce3+ Nanocrystalline Particles.

NaBiF4 nanocrystalline particles were synthesized by means of a facile precipitation synthesis route to explore upconversion emission properties when doped with lanthanide ions. In particular, the incorporation of the Yb3+-Ho3+-Ce3+ triad with controlled ion concentration facilitates near-IR pumping conversion into visible light, with the possibility of color emission tuning depending on Ce3+ doping amount. We observed that introducing a Ce3+ content up to 20 at.% in NaBiF4:Yb3+/Ho3+, the chromaticity progressively turns from green for the Ce3+ undoped system to red. This is due to cross-relaxation mechanisms between Ho3+ and Ce3+ ions that influence the relative efficiency of the overall upconversion pathways, as discussed on the basis of a theoretical rate equation model. Furthermore, experimental results suggest that the photoexcitation of intra-4f Ho3+ transitions with light near the UV-visible edge can promote downconverted Yb3+ near-IR emission through quantum cutting triggered by Ho3+-Yb3+ energy transfer mechanisms. The present study evidences the potentiality of the developed NaBiF4 particles for applications that exploit lanthanide-based light frequency conversion and multicolor emission tuning.

High-Speed Atomic Force Microscopy Reveals Spontaneous Nucleosome Sliding of H2A.Z at the Subsecond Time Scale.

Nucleosome dynamics, such as nucleosome sliding and DNA unwrapping, are important for gene regulation in eukaryotic chromatin. H2A.Z, a variant of histone H2A that is highly evolutionarily conserved, participates in gene regulation by forming unstable multipositioned nucleosomes in vivo and in vitro. However, the subsecond dynamics of this unstable nucleosome have not been directly visualized under physiological conditions. Here, we used high-speed atomic force microscopy (HS-AFM) to directly visualize the subsecond dynamics of human H2A.Z.1-nucleosomes. HS-AFM videos show nucleosome sliding along 4 nm of DNA within 0.3 s in any direction. This sliding was also visualized in an H2A.Z.1 mutant, in which the C-terminal half was replaced by the corresponding canonical H2A amino acids, indicating that the interaction between the N-terminal region of H2A.Z.1 and the DNA is responsible for nucleosome sliding. These results may reveal the relationship between nucleosome dynamics and gene regulation by histone H2A.Z.

Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy.

Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.

Macrocyclic Peptide-Conjugated Tip for Fast and Selective Molecular Recognition Imaging by High-Speed Atomic Force Microscopy.

Fast and selective recognition of molecules at the nanometer scale without labeling is a much desired but still challenging goal to achieve. Here, we show the use of high-speed atomic force microscopy (HS-AFM) for real-time and real-space recognition of unlabeled membrane receptors using tips conjugated with small synthetic macrocyclic peptides. The single-molecule recognition method is validated by experiments on the human hepatocyte growth factor receptor (hMET), which selectively binds to the macrocyclic peptide aMD4. By testing and comparing aMD4 synthesized with linkers of different lengths and rigidities, we maximize the interaction between the functionalized tip and hMET added to both a mica surface and supported lipid bilayers. Phase contrast imaging by HS-AFM enables us to discriminate nonlabeled hMET against the murine MET homologue, which does not bind to aMD4. Moreover, using ligands and linkers of small size, we achieve minimal deterioration of the spatial resolution in simultaneous topographic imaging. The versatility of macrocyclic peptides in detecting unlimited types of membrane receptors with high selectivity and the fast imaging by HS-AFM broaden the range of future applications of this method for molecular recognition without labeling.

Plasma membrane anchored nanosensor for quantifying endogenous production of H2O2 in living cells.

Hydrogen peroxide (H2O2) is one of the main second messengers involved in signaling pathways controlling cell metabolism. During tumorigenesis H2O2 is generated on the extracellular space by membrane-associated NADPH oxidases and superoxide dismutase to stimulate cell proliferation and preservation of the transformed state. Accordingly, a characteristic feature of malignant cells is overproduction of H2O2 in the extracellular milieu and the subsequent absorption in the cytosol. Since the most significant gradients of endogenous extracellular H2O2 can be observed only in a very shallow region of the fluid in contact with the plasma membrane, we show here the use of a newly designed nanosensor anchored to the outer cell surface and capable of quantifying H2O2 at nanometer distance from the membrane proteins responsible for its production. This biosensor is built upon gold nanoparticles functionalized with a H2O2-sensitive boronate compound that is probed using surface enhanced Raman spectroscopy (SERS). The highly localized information obtained on the cell surface by SERS analysis is combined with analytical methods of redox biology to estimate the associated levels of intracellular H2O2 responsible for cell signaling. The results obtained from A549 lung cancer cell line show localized spots on the cell surface at concentration up to 12 µM, associated to intracellular concentration up to 5.1 nM.

Bioconjugation strategy for cell surface labelling with gold nanostructures designed for highly localized pH measurement.

Regulation of intracellular pH is critically important for many cellular functions. The quantification of proton extrusion in different types of cells and physiological conditions is pivotal to fully elucidate the mechanisms of pH homeostasis. Here we show the use of gold nanoparticles (AuNP) to create a high spatial resolution sensor for measuring extracellular pH in proximity of the cell membrane. We test the sensor on HepG2 liver cancer cells and MKN28 gastric cancer cells before and after inhibition of Na+/H+ exchanger. The gold surface conjugation strategy is conceived with a twofold purpose: i) to anchor the AuNP to the membrane proteins and ii) to quantify the local pH from AuNP using surface enhanced Raman spectroscopy (SERS). The nanometer size of the cell membrane anchored sensor and the use of SERS enable us to visualize highly localized variation of pH induced by H+ extrusion, which is particularly upregulated in cancer cells.

Surface modifications and oxidative degradation in MPC-grafted highly cross-linked polyethylene liners retrieved from short-term total hip arthroplasty.

Highly cross-linked polyethylene (HXLPE) hip liners grafted with 2-methacryloyloxyethyl phosphorylcholine (MPC) on their bearing surfaces have recently been commercialized as components of a new generation of artificial hip joints, while improvements in wear resistance and biocompatibility were reported based on in vitro studies. The present study aimed at evaluating the surface modification and oxidative degradation in short-term retrieved MPC-grafted liners by X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FT-IR) with attenuated total reflection (ATR) equipment and Raman spectroscopy. In none of 3 samples of retrieved MPC-grafted liners, detectable MPC graft remained on the bearing surfaces although 2 samples yet contained remains of MPC polymer in their rim zone. These results revealed that the MPC polymer might have quickly disappeared from the bearing surface under in vivo loading, which is more severe than the in vitro one. Furthermore, a detectable oxidation index (OI) value (>0.1) was not only observed in any zone of any sample investigated, but also in the rim zones of Samples 1 and 2, which surprisingly experienced the most remarkable increase in OI value detected in this study. We thus confirmed that: (i) annealing of HXLPE cannot completely remove free radicals; (ii) the MPC graft has no beneficial effect in protecting HXLPE against oxidation and wear; and, (iii) lipid absorption occurred even in the rim zone where the MPC layer remained. Based on these evidences we consider that the declaimed advanced MPC technology is not a suitable one to elongate the in vivo lifetime of hip joints. STATEMENT OF SIGNIFICANCE: Several studies reported that highly crosslinked polyethylene (HXLPE) have resulted in reduced wear in total hip arthroplasty. Beyond those studies, HXLPE hip liners grafted with 2-methacryloyloxyethyl phosphorylcholine (MPC) on their bearing surface were extensively studied in vitro and then commercialized as a new generation of artificial hip joints. The present study reports for the first time results about the evaluation of surface modification and oxidative degradation in retrieved the MPC grafted liners. The findings of this investigation clearly show that the MPC layer has been peeled off on the bearing surface of the liner main wear zone although the MPC layer remained on the surface of the rim zones. Furthermore, we assessed the microstructural modifications and the oxidation drifts that occurred in vivo in the hip joints despite the presence of the MPC layer.

Raman micro-spectroscopy as a viable tool to monitor and estimate the ionic transport in epithelial cells.

The typical response to the lowering of plasma Na+ concentration and blood pressure in our body involves the release of aldosterone from the adrenal glands, which triggers the reabsorption of sodium in the kidney. Although the effects of aldosterone on this physiological mechanism were extensively studied in the past decades, there are still some aspects to be fully elucidated. In the present study, we propose for the first time a new approach based on Raman spectroscopy to monitor the ionic activity in aldosterone-treated A6 renal epithelial cells. This spectroscopic technique is capable of probing the cells through their thickness in a non-destructive and nimble way. The spectroscopic variations of the Raman bands associated to the O-H stretching of water were correlated to the variations of ionic concentration in the intracellular and extracellular fluids. The increase of Na+ concentration gradients was clearly visualized in the cytosol of aldosterone-treated cells. The enhancement of the Na+ current density induced by aldosterone was estimated from the variation of the ionic chemical potential across the intracellular space. In addition, the variation of the O-H Raman bands of water was used to quantify the cell thickness, which was not affected by aldosterone.

Effect of e-beam sterilization on the in vivo performance of conventional UHMWPE tibial plates for total knee arthroplasty.

Although the introduction of highly cross-linked polyethylene is effective in reducing the amount of wear, there are still major concerns regarding the use of this material in total knee arthroplasty (TKA), essentially due to the reduction of fatigue resistance and toughness. Monitoring the in vivo performance of different types of UHMWPE is a much needed task to tackle the lack of information on which should be the most reliable choice for TKA. The present study was aimed at investigating the mid-term degradation of electron beam sterilized conventional UHMWPE tibial plates. Visual inspection enabled to grade the surface damage of 12 retrievals according to the Hood's score: the total wear damage correlates to the in vivo time (Spearman's ρ=0.681, p<0.05) and BMI (ρ=0.834, p<0.001). Surface degradation was less severe than that quantified in similar studies on γ-sterilized UHMWPE. Raman and infra-red spectroscopies were utilized to unfold the microstructural modifications. In the load zone, polyethylene whitened damage regions were noticed in the inserts implanted longer than 1year, in which oxidation index (OI) is clearly higher than 1 (max 8). The maximum OI (ρ=0.802, p<0.005) and αc (ρ=0.816, p<0.005) correlate to the implantation time in the load zone. The crystallinity increased along with the extent of oxidation. Concentration of absorbed species from synovial fluid is higher in the contact zone and correlates to maximum OI (Spearman's ρ=0.699, p=0.011). Absorption was promoted in the contact area by the mechanical action of the femoral counterpart and it exacerbated the oxidative degradation in retrievals with high concentration of absorbed species. In the non-load zone, mild but detectable oxidation was observed, probably due to free radicals trapped after sterilization. STATEMENT OF SIGNIFICANCE: Although several clinical studies on retrieved tibial bearings have been published so far, monitoring and comparing the in vivo performance of different types of UHMWPE is still a much needed task. The present study reports for the first time results on the effect of sterilization by electron beam on the mid-term in vivo performance of conventional UHMWPE tibial plates. In the present investigation, visual inspection of wear damage based on the Hood's scoring method, Raman micro-spectroscopy and Fourier-transformed infrared spectroscopy were utilized to unveil the damage, the microstructural modifications and the oxidation occurred during implantation. The findings of this investigation have been discussed and compared to previous clinical studies on γ-air sterilized, γ-inert sterilized tibial bearings.

On the molecular interaction between femoral heads and polyethylene liners in artificial hip joints: phenomenology and molecular scale phenomena.

The chemical interaction of femoral heads with polyethylene liners is an important new area of research in total hip arthroplasty (THA) as a consequence of an unabating quest for elongated artificial joint lifetimes. It is a topic that goes beyond simple mechanical behavior or wear. It seeks to describe the prosthetic device as a whole by adding chemical considerations to mechanics and abrasion. One of the poorly understood chemical interactions is oxygen affinity of the femoral heads. While oxygen unavoidably diffuses into the non-crystalline enclaves of the polyethylene liner as a result of its contact with the tribolayer, a ceramic femoral head can either attract or release oxygen due to its tribochemical interactions. In this study, changes in liner crystallinity and oxidation were observed during simple static contact with different femoral heads within a hydrothermal environment. Examinations were made using Raman microprobe spectroscopy coupled with nanometer-scale spectroscopic analyses (x-ray photoelectron spectroscopy (XPS) and cathodoluminescence (CL) spectroscopy) on different sets of ceramic and polyethylene couples exposed for increasing periods to a hydrothermal environment. Even in the absence of frictional loading, structural modifications were clearly detected, statistically validated, and correlated with the oxygen affinity of various femoral head materials. It was found that oxide (Al2O3, Al2O3/ZrO2, and m-ZrO2) and non-oxide (Si3N4) ceramic heads release or attract nascent oxygen species, respectively. This research unequivocally identified a new gap in our knowledge of THA biomaterial interactions, with future studies and material improvements undoubtedly leading to an increase in longevity for prosthetic joints.

Silicon Nitride: A Synthetic Mineral for Vertebrate Biology.

The remarkable stoichiometric flexibility of hydroxyapatite (HAp) enables the formation of a variety of charged structural sites at the material's surface which facilitates bone remodeling due to binding of biomolecule moieties in zwitterionic fashion. In this paper, we report for the first time that an optimized biomedical grade silicon nitride (Si3N4) demonstrated cell adhesion and improved osteoconductivity comparable to highly defective, non-stoichiometric natural hydroxyapatite. Si3N4's zwitterionic-like behavior is a function of the dualism between positive and negative charged off-stoichiometric sites (i.e., N-vacancies versus silanols groups, respectively). Lattice defects at the biomaterial's surface greatly promote interaction with positively- and negatively-charged functional groups in biomolecules, and result in the biologically effective characteristics of silicon nitride. These findings are anticipated to be a starting point for further discoveries of therapeutic bone-graft substitute materials.

Validation of a protocol based on Raman and infrared spectroscopies to nondestructively estimate the oxidative degradation of UHMWPE used in total joint arthroplasty.

UNLABELLED: As a matter of fact, the in vivo oxidative degradation of highly cross-linked polyethylene (HXLPE) still remains one of the limiting factors that affect the long term survivorship of joint replacements. Recent studies clearly pointed out that also the new generation of highly cross-linked and remelted polyethylene components in total hip and knee replacement underwent unexpected oxidation after 5-10years of implantation. The standard methodology to investigate the oxidation of polyethylene (PE) relies on the use of infrared spectroscopy, which, if from one hand is a reliable technique for the detection of oxidized species containing carbonyl group, on the other hand it is not capable of discriminating the fraction of carboxyl acids that is responsible for chain scission and subsequent deterioration of the mechanical properties of the polymer. In the present study we validate a new protocol based on Raman spectroscopy, which is suitable on assessing the structural degradation of polyethylene induced by oxidation. Following in vitro accelerated aging experiments, the oxidation index (OI) of different commercially available HXLPEs, as calculated by infrared spectroscopy according to ASTM standard, has been univocally correlated to the most severe variation of crystalline phase (αc), as calculated by Raman spectroscopy. In each material, locations with equal values of OI showed different degree of recrystallization induced by chain scission, confirming that infrared spectroscopy might overestimate the effective mechanical degradation of the polymer. In addition, as compared to the standards based on infrared spectroscopy, this new method of assessing oxidation enables to investigate the degradation occurring on the original surface of HXLPE components, due to the nondestructive nature of Raman spectroscopy and its high spatial resolution. STATEMENT OF SIGNIFICANCE: In the present study we validate a new protocol based on Raman spectroscopy, which is suitable on assessing the structural degradation of polyethylene induced by oxidation. In fact, the standard methodology to investigate the oxidation in polyethylene relies on the use of infrared spectroscopy, which is capable of detecting the presence of oxidized species containing carbonyl group, the main products of oxidation in polyolefins. If from one hand this technique enables quantitative analysis of oxidation, on the other hand it is not capable of discriminating the fraction of species with carbonyl groups responsible for the chain scission. In fact, esters, ketones and carboxyl acids are products of oxidation with carbonyl groups commonly formed on polyethylene at the end of the oxidative cascade initiated by the presence of free radicals, but only the latter are responsible for the chain scission and the subsequent deterioration of the mechanical properties. The oxidation index as obtained according to the ASTM standards is not univocally correlated to a certain degree of mechanical deterioration, but, in simple words, two retrievals with the same amount of carbonyl groups might have had different degradation of the mechanical properties. Recrystallization is a direct consequence of the reduction of molecular weight that occurs after chain scission. Raman spectroscopy (RS) is a viable non-destructive method to assess the fraction of crystalline phase in polyethylene and, due to its high spatial resolution, is perfectly suitable to analyze the microstructural modification at the mesoscopic scale, where the effects of oxidation manifest themselves. The aim of the present paper is twofold: i) to compare the microstructural modifications caused by in vitro oxidation on 5 different types of polyethylene currently available on the market of joint replacements; ii) to establish a protocol based on the comparative analysis of IR and RS results to obtain a phenomenological correlation capable to judge the mechanical deterioration of the material induced by the oxidative degradation.

Silicon Nitride Bioceramics Induce Chemically Driven Lysis in Porphyromonas gingivalis.

Organisms of Gram-negative phylum bacteroidetes, Porphyromonas gingivalis, underwent lysis on polished surfaces of silicon nitride (Si3N4) bioceramics. The antibacterial activity of Si3N4 was mainly the result of chemically driven principles. The lytic activity, although not osmotic in nature, was related to the peculiar pH-dependent surface chemistry of Si3N4. A buffering effect via the formation of ammonium ions (NH4(+)) (and their modifications) was experimentally observed by pH microscopy. Lysis was confirmed by conventional fluorescence spectroscopy, and the bacteria's metabolism was traced with the aid of in situ Raman microprobe spectroscopy. This latter technique revealed the formation of peroxynitrite within the bacterium itself. Degradation of the bacteria's nucleic acid, drastic reduction in phenilalanine, and reduction of lipid concentration were observed due to short-term exposure (6 days) to Si3N4. Altering the surface chemistry of Si3N4 by either chemical etching or thermal oxidation influenced peroxynitrite formation and affected bacteria metabolism in different ways. Exploiting the peculiar surface chemistry of Si3N4 bioceramics could be helpful in counteracting Porphyromonas gingivalis in an alkaline pH environment.

Surface modifications induced by in-vitro wear and oxidation on γ-irradiated UHMWPE hip liners belonging to different commercial generations.

Single-step and three-step irradiated and annealed ultra-high molecular weight polyethylene (UHMWPE) hip liners have been studied by means of Raman spectroscopy (RS) and Fourier transform infrared spectroscopy (FT-IR), in order to clarify the microstructural modifications induced by in vitro oxidative degradation and wear. These spectroscopic techniques enabled us to measure profiles of oxidation index (OI), crystalline (αc), amorphous (αa), and third phase (αb) fractions along the subsurface of the acetabular cups as a function of in vitro oxidation time or after standard testing in hip simulator. Microtomed sections of the liners after accelerated aging (ASTM F2003-02) showed that oxidation profiles developed differently during the first two weeks, while all samples aged longer than 2 weeks revealed OI increasing with lower rates. The initial oxidation of the single-step-annealed material was higher than the one retrieved from the 3-step-annealed material and showed a peak of OI located at a depth of ~1mm below the exposed surface. The profiles of αc, calculated from the same sample cross-sections, followed trends similar to the respective OI profiles, which enabled a phenomenological (but quantitative) correlation between oxidation and crystallization processes to be obtained. Wear simulation under edge loading conditions was conducted on series of four samples of the above two types of irradiated and annealed materials, and for two different liner thicknesses (5.9 and 7.9 mm). The wear rates calculated at the end of the test were very low for all samples (max 2.08 mm(3)/mc for the thinner liners of the single-step irradiated and annealed material). The results indicated that there was a statistically significant increase in both wear rate and volume loss only for the thinner single-step irradiated and annealed liners. Surface analyses by Raman spectroscopy revealed distinct gradients of crystallinity, amorphous, and third phase fractions along the in-depth axis. In both types of UHMWPE materials, the worn area showed an increase of crystallinity at the expense of the third phase. Differences in crystallinity profiles observed at the wear zone of liners with different thicknesses were correlated to the higher contact stress experienced by thinner liners.

In-vivo degradation of middle-term highly cross-linked and remelted polyethylene cups: Modification induced by creep, wear and oxidation.

In this study Raman (RS) and Fourier Transform Infrared (FT-IR) spectroscopic techniques were exploited to study 11 retrieved liners made of remelted highly cross-linked polyethylene (HXLPE), with the intent to elucidate their in-vivo mechanical and chemical degradation. The retrievals had different follow-ups, ranging from a few months to 7 years of implantation time and belong to the first generation of highly cross-linked and remelted polyethylene clinically introduced in 1999, but still currently implanted. Raman assessments enabled to discriminate contributes of wear and creep on the total reduction of thickness in different locations of the cup. According to our results, although the most of the viscoelastic deformation occurred during the first year (bedding-in period), it progressed during the steady wear state up to 7 years with much lower but not negligible rate. Overall, the wear rate of this remelted HXLPE liner was low. Preliminary analysis on microtomed sections of the liners after in-vivo and in-vitro accelerated aging (ASTM F2003-02) enabled to obtain a phenomenological correlation between the oxidation index (OI) and the amount of orthorhombic phase fraction (αc), which can be easily non-destructively measured by RS. Profiles of αc obtained from different locations of the cups were used to judge the oxidative degradation of the 11 retrievals, considering also the ex-vivo time elapsed from the revision surgery to the spectroscopic experiments. Low but measurable level of oxidation was detected in all the short-term retrievals, while in the middle-term samples peaks of OI were observed in the subsurface (up to OI=4.5), presumably induced by the combined effect of mechanical stress, lipid absorption and prolonged ex-vivo shelf-aging in air.