3D Electrospun Polycaprolactone Scaffolds to Assess Human Periodontal Ligament Cells Mechanobiological Behaviour.

While periodontal ligament cells are sensitive to their 3D biomechanical environment, only a few 3D in vitro models have been used to investigate the periodontal cells mechanobiological behavior. The objective of the current study was to assess the capability of a 3D fibrous scaffold to transmit a mechanical loading to the periodontal ligament cells. Three-dimensional fibrous polycaprolactone (PCL) scaffolds were synthetized through electrospinning. Scaffolds seeded with human periodontal cells (103 mL-1) were subjected to static (n = 9) or to a sinusoidal axial compressive loading in an in-house bioreactor (n = 9). At the end of the culture, the dynamic loading seemed to have an influence on the cells' morphology, with a lower number of visible cells on the scaffolds surface and a lower expression of actin filament. Furthermore, the dynamic loading presented a tendency to decrease the Alkaline Phosphatase activity and the production of Interleukin-6 while these two biomolecular markers were increased after 21 days of static culture. Together, these results showed that load transmission is occurring in the 3D electrospun PCL fibrous scaffolds, suggesting that it can be used to better understand the periodontal ligament cells mechanobiology. The current study shows a relevant way to investigate periodontal mechanobiology using 3D fibrous scaffolds.

Morphological and Mechanical Characterization of Extracellular Vesicles and Parent Human Synoviocytes under Physiological and Inflammatory Conditions.

The morphology of fibroblast-like synoviocytes (FLS) issued from the synovial fluid (SF) of patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), or from healthy subjects (H), as well as the ultrastructure and mechanical properties of the FLS-secreted extracellular vesicles (EV), were analyzed by confocal microscopy, transmission electron microscopy, atomic force microscopy, and tribological tests. EV released under healthy conditions were constituted of several lipid bilayers surrounding a viscous inner core. This "gel-in" vesicular structure ensured high mechanical resistance of single vesicles and good tribological properties of the lubricant. RA, and to a lesser extent OA, synovial vesicles had altered morphology, corresponding to a "gel-out" situation with vesicles surrounded by a viscous gel, poor mechanical resistance, and poor lubricating qualities. When subjected to inflammatory conditions, healthy cells developed phenotypes similar to that of RA samples, which reinforces the importance of inflammatory processes in the loss of lubricating properties of SF.

Synovial Extracellular Vesicles: Structure and Role in Synovial Fluid Tribological Performances.

The quality of the lubricant between cartilaginous joint surfaces impacts the joint's mechanistic properties. In this study, we define the biochemical, ultrastructural, and tribological signatures of synovial fluids (SF) from patients with degenerative (osteoarthritis-OA) or inflammatory (rheumatoid arthritis-RA) joint pathologies in comparison with SF from healthy subjects. Phospholipid (PL) concentration in SF increased in pathological contexts, but the proportion PL relative to the overall lipids decreased. Subtle changes in PL chain composition were attributed to the inflammatory state. Transmission electron microscopy showed the occurrence of large multilamellar synovial extracellular vesicles (EV) filled with glycoprotein gel in healthy subjects. Synovial extracellular vesicle structure was altered in SF from OA and RA patients. RA samples systematically showed lower viscosity than healthy samples under a hydrodynamic lubricating regimen whereas OA samples showed higher viscosity. In turn, under a boundary regimen, cartilage surfaces in both pathological situations showed high wear and friction coefficients. Thus, we found a difference in the biochemical, tribological, and ultrastructural properties of synovial fluid in healthy people and patients with osteoarthritis and arthritis of the joints, and that large, multilamellar vesicles are essential for good boundary lubrication by ensuring a ball-bearing effect and limiting the destruction of lipid layers at the cartilage surface.

Nano/micro implant debris affect osteogenesis by chondrocytes: Comparison between ceramic and UHMWPE from hip walking simulator.

A new generation of ceramic on ceramic (BIOLOX ®delta) bearings has emerged more than 10 years ago proving a high resistance to wear and good clinical results. However, biological reactions to wear debris, particularly the nanoparticles, need to be evaluated. The first originality of this study is to start from real wear particles obtained by the hip walking simulator (CERsim). These particles were compared with particles obtained by usual methods to assess the biocompatibility of materials: press machine (CERpress). Two ranges of ceramic particles were thus observed: ceramic particles with micron (intergranular fractures) and nano sizes (intragranular fractures), and characterized compared to ultra-high molecular weight polyethylene (UHMWPE). The second originality of this work is to assess the cellular reaction using the primary joint chondrocyte cultures simulating the osteogenesis process and not the cell lines, which are used to simulate the biological reaction of osteolysis. The first results showed a significant difference in cell viability between the cells in contact with particles from the walking simulator and those obtained with the press machine. On the other hand, it was found that the way of extraction of the particles from the lubricant could significantly affect the biological reaction. More interestingly, nano-sized ceramic particles showed a significant impact on the secretion of functional inflammatory mediators, agreeing with recent results in vivo. These novel methods of characterizing the osteogenic impact of UHMWPE and ceramic wear debris can complement the conventional expertise method focusing previously on the osteolysis aspect.

Metal-Metal Hip Prosthesis and Kidney Cancer: Assumed Role of Chromium and Cobalt Overload.

BACKGROUND The case of a patient with bilateral renal cancers diagnosed at 94 and 120 months after metal-on-metal hip placement may serve as a warning. It suggests that there may be a need for kidney echography observation of patients with similar types of prostheses. CASE REPORT A 61-year-old woman received a metal-on-metal hip prosthesis for degenerative arthritis in January 2007. In November 2014, after bleeding from the renal tract, she was diagnosed with clear cell carcinoma of the right kidney. When she returned to her orthopaedic surgeon 1 year later, a blood test showed a serum cobalt level that exceeded the French medical agency recommendation. After the patient's metallic acetabulum was replaced in September 2015, her blood cobalt level fell. However, in February 2017, she was diagnosed with adenocarcinoma of the left kidney. Laser-induced breakdown spectroscopy (LIBS) showed cobalt, chromium, and silica overload in both the patient's kidneys despite the drop in serum levels. CONCLUSIONS In this case, exposure to a cobalt-chromium implant with high particulate wear, LIBS results showing chromium overload of the kidneys, diagnosis of renal cancer at 7 years, 10 months and 10 years in a patient with a metal-on-metal hip prosthesis suggests that there may be a causal relationship between the implant, carcinogenic chromium intoxication, and development of renal cancer.

Identification of inorganic particles resulting from degradation of ESSURE® implants: Study of 10 cases.

OBJECTIVE: Approximately 750,000 women worldwide have undergone ESSURE hysteroscopic sterilization since 2002. In 2015, an increase in adverse effects was noted, with gynaecological and systemic symptoms reported. Scanning electron microscopy (SEM) analysis of fallopian tube and uterine horn tissues and implants, after hysterectomy or salpingectomy, revealed the presence of inorganic particles resulting from implant degradation. STUDY DESIGN: Ten patients (age 42-53 years) were included in this study. Of these, eight patients had undergone hysterectomy and two patients had undergone salpingectomy. Mean exposure time was 85.5 months (standard deviation 26.8 months, range 34-105 months). Mineralogical analyses were performed on 13 tissue biopsies and four implants by SEM coupled with energy dispersive x-ray spectrometry. RESULTS: In five of the 10 patients, tin particles were observed in fallopian tube or uterine horn tissues with inflammatory cell reactions. In the other five cases, iron, chromium, nickel or platinum particles were observed. For implants, major deterioration of the weld zone was observed with either destroyed appearance or the presence of an organic coating containing numerous particles. DISCUSSION AND CONCLUSION: Analysis of the preclinical studies performed by the manufacturer suggests that degradation of the tin weld plays a major role in these adverse events, with increasing leaching and corrosion between 3 and 6 months for an intratubal insert that si designed to remain in an woman's body for her entire life. For patients with gynaecological symptoms (e.g. pain, metrorragies) needing explantation, these findings raise the question of a causal relationship between tin particles from implant degradation and the inflammatory tissue response. For patients with systemic symptoms (e.g. blurred vision, headache, asthenia, myalgia), the hypothesis that these symptoms may be related to the formation of organotin (chemical compounds based on tin with hydrocarbon substituents) in the body has yet to be proven. Tin levels in blood have to be measured before and after explantation. To the authors' knowledge, this is the first study to report significant degradation of the ESSURE implant weld, evidenced by the detection of tin particles in the uterine tissue of patients and comparison of the welding zone between unused and used implants.

Membrane Fluidity as a New Means to Selectively Target Cancer Cells with Fusogenic Lipid Carriers.

Lipid-based carriers such as liposomes represent one of the most advanced classes of drug delivery systems. Their clinical success relies on their composition, similar to that of the cell membrane. Their cellular specificity often relies on a ligand-receptor interaction. Although differences in the physicochemical properties of the cell membrane between tumor and nontumor cells have been reported, they are not systematically used for drug delivery purposes. In this report, a new approach was developed to ensure selective targeting based on physical compatibility between the target and the carrier membranes. By modulating the liposome composition and thus its membrane fluidity, we achieved selective targeting on four cancer cell lines of varying aggressiveness. Furthermore, using membrane-embedded and inner core-encapsulated fluorophores, we assessed the mechanism of this interaction to be based on the fusion of the liposome with the cell membranes. Membrane fluidity is therefore a major parameter to be considered when designing lipid drug carriers as a promising, lower cost alternative to current targeting strategies based on covalent grafting.

Phospholipid Vesicles in Media for Tribological Studies against Live Cartilage.

INTRODUCTION: Pre-clinical testing of hemiarthroplasty devices requires that the tribological conditions present in vivo with live cartilage be closely duplicated. A current limitation in the tribological testing of live cartilage involves the use of cell-culture media as lubricant. STUDY AIM: to develop and test a new hyaluronan-phospholipid based medium (HA-phospholipid medium) that combines the rheological and frictional properties of synovial fluid with the nourishing properties of culture media to keep cells alive. MATERIALS AND METHODS: The HA-phospholipid medium consisted of culture medium with added phospholipid dipalmitoylphosphatidylcholine (0.3 mg/mL), and hyaluronic acid (2.42 mg/mL). A standard cell culture medium was used as the control. The rheology of each medium was determined using a flat plate configuration. Bovine calf cartilage was used to assess cell viability and friction in each medium. For friction measurements, a cobalt-chrome alloy ball was articulated against cartilage disks immersed in medium. RESULTS: Lipid vesicles 0.1 to 50 µm in diameter were identified in the HA-phospholipid medium. Cartilage cell viability was significantly higher in the HA-phospholipid medium (62% ± 8%, 95% CI) than in control medium (49.5% ± 5%) (p = 0.009). The HA-phospholipid medium exhibited strong shear-thinning behavior, similar to synovial fluid, with viscosities ~100-fold higher at 10 s-1 and 5-fold higher at 20,000 s-1 than the approximately Newtonian control medium. The HA-phospholipid medium also yielded 20% lower friction values than the control medium after one hour of testing. CONCLUSIONS: The rheological and friction results indicate that the HA-phospholipid medium is superior to the control cell culture medium in emulating the shear thinning and lubricative properties of natural synovial fluid, making it more clinically relevant for in vitro wear and friction testing with live cartilage.

Ultrastructural analysis of healthy synovial fluids in three mammalian species.

A better knowledge of synovial fluid (SF) ultrastructure is required to further understand normal joint lubrication and metabolism. The aim of the present study was to elucidate SF structural features in healthy joints from three mammalian species of different size compared with features in biomimetic SF. High-resolution structural analysis was performed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and environmental SEM/wet scanning transmission electron microscopy mode complemented by TEM and SEM cryogenic methods. Laser-scanning confocal microscopy (LCM) was used to locate the main components of SF with respect to its ultrastructural organization. The present study showed that the ultrastructure of healthy SF is built from a network of vesicles with a size range from 100 to a few hundred nanometers. A multilayered organization of the vesicle membranes was observed with a thickness of about 5 nm. LCM study of biological SF compared with synthetic SF showed that the microvesicles consist of a lipid-based membrane enveloping a glycoprotein gel. Thus, healthy SF has a discontinuous ultrastructure based on a complex network of microvesicles. This finding offers novel perspectives for the diagnosis and treatment of synovial joint diseases.

Multiscale characterization of partially demineralized superficial and deep dentin surfaces.

The objective of this study was to address the following question: 'Which properties are modified in partially demineralized surfaces, compared with non-demineralized dentin surfaces, following orthophosphoric acid-etching as performed in clinical procedures?'. For this purpose, the complementary techniques atomic force microscopy/spectroscopy, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and contact angle measurements were used to provide a multiscale characterization of the dentin substrate undergoing the acidic preconditioning designed to enhance wetting. Special attention was given to the influence of the etching pretreatment on the nanomechanical properties at different levels of dentin surfaces, in both dry and hydrated conditions. The four-sided pyramid model (extended Hertz contact model) proved to be accurate for calculating the apparent Young's modulus, offering new information on the elasticity of dentin. The modulus value notably decreased following etching and surface hydration. This study underlines that after the acid etching pretreatment the contribution of the nanomechanical, morphological, and physicochemical modifications has a strong influence on the dentin adhesion properties and thus plays a significant role in the coupling of the adhesive-resin composite build-up material at the dentin surface.

Alteration of cartilage mechanical properties in absence of ß1 integrins revealed by rheometry and FRAP analyses.

CONTEXT: Mechanical properties are essential for biological functions of the hyaline cartilage such as energy dissipation and diffusion of solutes. Mechanical properties are primarily dependent on the hierarchical organization of the two major extracellular matrix (ECM) macromolecular components of the cartilage: the fibrillar collagen network and the glycosaminoglycan (GAG)-substituted proteoglycan, mainly aggrecan, aggregates. Interaction of chondrocytes, the only cell type in the tissue, with the ECM through adhesion receptors is involved in establishing mechanical stability via bidirectional transduction of both mechanical forces and chemical signals. In this study, we aimed to determine the role of the transmembrane ß1 integrin adhesion receptors in cartilage biomechanical properties by the use of genetic modification in mice. METHODS: Costal cartilages of wild type and mutant mice lacking ß1 integrins in chondrocytes were investigated. Cartilage compressive properties and solute diffusion were characterized by rheometric analysis and Fluorescence Recovery After Photobleaching (FRAP), respectively. Cartilage tissue sections were analyzed by histology, immunohistochemistry and transmission electron microscopy (TEM). RESULTS: At the histological level, the mutant costal cartilage was characterized by chondrocyte rounding and loss of tissue polarity. Immunohistochemistry and safranin orange staining demonstrated apparently normal aggrecan and GAG levels, respectively. Antibody staining for collagen II and TEM showed comparable expression and organization of the collagen fibrils between mutant and control cartilages. Despite the lack of gross histological and ultrastructural abnormalities, rheological measurements revealed that the peak elastic modulus in compression of mutant cartilage was 1.6-fold higher than the peak elastic modulus of wild-type sample. Interestingly, the diffusion coefficient within the mutant cartilage tissue was found to be 1.2-fold lower in the extracellular space and 14-fold lower in the pericellular (PCM) space compared to control. CONCLUSION: The results demonstrate that the absence of ß1 integrins on the surface of chondrocytes increases the stiffness and modifies the diffusion properties of costal cartilage. Our data imply that ß1 integrins-mediated chondrocyte-matrix interactions directly affect cartilage biomechanics probably by modifying physical properties of individual cells. This study thus highlights the crucial role of ß1 integrins in the cartilage function.

Nanomechanical and tribological characterization of the MPC phospholipid polymer photografted onto rough polyethylene implants.

Grafting biomimetic polymers onto biomaterials such as implants is one of the promising approaches to increase their tribological performance and biocompatibility and to reduce wear. In this paper, poly(2-methacryloyloxyethyl phosphorylcholine) (p(MPC)) brushes were obtained by photografting MPC from the rough surface of ultra high molecular weight polyethylene (UHMWPE) joint implants. Such substrates have a high roughness (Ra∼650nm) which often has the same order of magnitude as the brush thickness, so it is very difficult to estimate the vertical density profile of the grafted content. The quality of the p(MPC) grafting was evaluated through a wide range of characterization techniques to reveal the effectiveness of the grafting: atomic force microcopy (AFM) imaging and force spectroscopy, contact angle, SEM/EDX, and confocal microscopy. After testing the methods on smooth glass substrate as reference, AFM nano-indentation proves to be a reliable non destructive method to characterize the thickness and the mechanical properties of the p(MPC) layer in liquid physiological medium. Tribological measurements using a homemade biotribometer confirm that, despite heterogeneity thickness (h=0.5-6µm), the p(MPC) layer covers the roughness of the UHMWPE substrate and acts as an efficient lubricant with low friction coefficient and no wear for 9h of friction.

Tenascin-X increases the stiffness of collagen gels without affecting fibrillogenesis.

Tenascin-X is an extracellular matrix protein whose absence leads to an Ehlers-Danlos Syndrome in humans, mainly characterised by connective tissue defects including the disorganisation of fibrillar networks, a reduced collagen deposition, and modifications in the mechanical properties of dense tissues. Here we tested the effect of tenascin-X on in vitro collagen fibril formation. We observed that the main parameters of fibrillogenesis were unchanged, and that the diameter of fibrils was not significantly different when they were formed in the presence of tenascin-X. Interestingly, mechanical analysis of collagen gels showed an increased compressive resistance of the gels containing tenascin-X, indicating that this protein might be directly involved in determining the mechanical properties of collagen-rich tissues in vivo.

Role of nanomechanical properties in the tribological performance of phospholipid biomimetic surfaces.

The role of phospholipid bilayers in controlling and reducing frictional forces between biological surfaces is investigated by three complementary experiments: friction forces are measured using a homemade tribometer, mechanical resistance to indentation is measured by AFM, and lipid bilayer degradation is controlled in situ during friction testing using fluorescence microscopy. DPPC lipid bilayers in the solid phase generate friction coefficients as low as 0.002 (comparable to that found for cartilage) that are stable through time. DOPC bilayers formed by the vesicle fusion method or the adsorption of mixed micelles generate higher friction coefficients. These coefficients increased through time, during which the bilayers degraded. The friction coefficient is correlated with the force needed to penetrate the bilayer with the AFM tip. With only one bilayer in the contact region, the friction increased to a similar value of about 0.08 for the DPPC and DOPC. Our study therefore shows that good mechanical stability of the bilayers is essential and suggests that the low friction coefficient is ensured by the hydration layers between adjacent lipid bilayers.