Attempts to eradicate two Pelargonium viruses (PFBV and PLPV) by meristem culture and shoot-tip cryotherapy

Attempts to eradicate the Pelargonium flower break virus (PFBV) and Pelargonium line pattern virus (PLPV) by meristem culture and apex “droplet-vitrification” cryopreservation was carried out using 5 different cultivars. A simple meristem culture did not permit to eliminate PFBV and only 15% of Pelargonium x hortorum ‘Stellar Artic’ plants regenerated from meristems was PLPV-ELISA-negative. Plants regenerated from cryopreserved apices were tested by DAS-ELISA after a 3-month growing period. Viruses were not detected in 25 and 50% of the tested plants for PFBV and PLPV respectively. Immunolocalisations were carried out for virus localisation in apices from greenhouse plants (control) and vitroplants regenerated after meristem culture or cryopreservation. Immunolocalisations realised on control explants excised from DAS-ELISA positive plants showed that PFBV and PLPV were present in the apices, even in the meristematic dome. However, viral particles were more numerous in the cells of the basal zone than in the more meristematic ones. Immunolocalisations realised on apices from the DAS-ELISA negative cryoregenerated plants showed the viruses were still present. Our results firstly demonstrated that PFBV and PLPV are even present inside meristematic cells and secondly that cryopreservation could decrease their amount in Pelargonium plants but without eliminating them totally. More knowledge on virus behaviour during cryopreservation processes could optimize the management of genetic resources using this conservation method

Nouvelle stratégie antibiofilm par dépôt LBL d’un polyélectrolyte cationique sur la membrane de dialyse anionique AN69

Cette étude présente une stratégie antibiofilm appliquée à une membrane de dialyse, l’AN69; il s’agit de réaliser une modification initiale de la surface de la membrane par un polyélectrolyte cationique, le poly(diallyldiméthyl-ammonium), noté PDADMA, selon un protocole de type « layer-by-layer ». Les caractéristiques physico-chimiques des deux membranes, l’AN69 et l’AN69 modifiée par le PDADMA, sont suivies par la détermination de la modification de la charge de la membrane en mettant en oeuvre des mesures de potentiels d’écoulement et de nombres de transports de Li+, de perméabilité hydraulique et des analyses morphologiques et topographiques effectuées par les microscopies électroniques à balayage et à force atomique, respectivement avant et après exposition à E. coli et à un biofilm marin. Nos résultats montrent tout d’abord un rôle majeur joué par l’attraction électrostatique entre les microorganismes et l’AN69 modifiée par le PDADMA à l’origine d’une adhésion forte des bactéries.Par ailleurs, nous avons mis au point un protocole original d’élimination du biofilm marin. Ce protocole consiste à immerger la membrane modifiée par le PDADMA et encrassée, dans une solution de chlorure de sodium 2M afin d’écranter les charges électrostatiques à l’origine de l’accroche du polyélectrolyte et permettre le décrochage du PDADMA qui entraîne avec lui l’encrassement (constitué de bactéries et autres résidus de biofilm). La recharge d’un film frais de PDADMA permet alors une réutilisation à l’infini de la membrane AN69. La simplicité de ce protocole « de régénération » ouvre la possibilité d’une modification non permanente des membranes de dialyse, dans le but de limiter les problèmes récurrents de biocolmatage et d’augmenter les durées de vie des membranes en milieu marin.The harmful effects generated by biofilms have an extremely high cost. In this study, we were interested in the formation of marine biofilms forming on the wellknown anionic dialysis membrane AN69 used in the reduction of seawater salinity for the preparation of body-washing solutions. We focused our study on two membranes: AN69 and AN69 modified (denoted AN69 mod) by the sorption of poly(diallyldimethyl-ammonium), a cationic polyelectrolyte denoted by PDADMA. Physico-chemical characteristics (membrane charge from Li+ transport and streaming potential measurements, water flux from hydraulic permeability measurements and morphological/topographical measurements using SEM and AFM, respectively) were monitered during the exposure of both membranes to E. coli and natural marine biofouling solutions. Our results showed that the modification of the AN69 membrane by PDADMA its charge and increased its electrostatic affinity for bacteria. Electrostatics forces are, therefore, the main forces responsible for membrane biofouling. Furthermore we developed a simple antibiofilm protocol based on a one-step dipping operation applied to the fouled AN69 membrane which eliminates the PDADMA and associated bacteria. For the first time, all deposited marine bacteria were easily eliminated allowing for the regeneration and sustainable use of the AN69 membrane

Chemical structure of methylmethacrylate-2-[2′,3′,5′-triiodobenzoyl]oxoethyl methacrylate copolymer, radio-opacity, in vitro and in vivo biocompatibility

The properties of copolymers (physical, chemical, biocompatibility, etc.) depend on their chemical structure and microstructural characteristics. We have prepared radio-opaque polymers based on the copolymers of methyl methacrylate (MMA) and 2-[2′,3′,5′-triiodobenzoyl]oxoethyl methacrylate (TIBOM). The copolymerization reaction between TIBOM and MMA showed that the reactivity ratios were r1 = 0.00029 and r2 = 1.2146. The composition diagram is typical for a practically non-homopolymerizable monomer (TIBOM) and a very reactive monomer (MMA). The copolymers were analyzed on an X-ray microcomputed tomograph and they proved to be radio-opaque even at low concentrations of TIBOM. The biocompatibility was tested both in vitro (with J774.2 macrophage and SaOS-2 osteoblast like cells) and in vivo in the rat. These materials were found to be non-toxic and were well tolerated by the organism. These combined results led to the suggestion that this type of polymer could be used as dental or bone cements in place of barium or zirconium particles, which are usually added to provide X-ray opacity

In vitro assessment of osteoblast and macrophage mobility in presence of β-TCP particles by videomicroscopy

β-TCP is widely used to repair bone defects due to its good biocompatibility, macroporosity (favoring bone ingrowth) and bioresorbability. However, cell interactions with the biomaterial at the first times of implantation remain largely unknown. We have observed cell behaviors in direct contact with β-TCP particles using long-term culture under videomicroscopy. Osteoblastlike cells (SaOs-2) and macrophages (J774.2 and mouse peritoneal macrophages) were cultured in the presence of β-TCP particles. For each experiment, images from 20 independent fields were acquired and stored every 15 min during 8 days. At the end of the culture, they were combined to generate time lapse videos; coverslips were fixed and observed by scanning electron microscopy (SEM). SaOs-2 proliferation was determined by counting cells on six different and independent fields at days 1, 3, and 6. Videos showed the capacity of cells to displace the particles. Dynamic follow-up showed active proliferation of SaOs-2 occurring in the direction of the particles. J774.2 and peritoneal macrophages did not proliferate but came in direct contact with the particles and actively eroded them. SEM showed that cells were stretched and fixed onto the surface and seemed to climb from the coverslip to the particles. The long-term culture under videomicroscopy allowed a better understanding of the colonization process of β-TCP particles by osteoblastlike cells and macrophages. Data obtained from long-term videomicroscopy are in agreement with in vivo observations confirming the interest of β-TCP to promote osteogenesis

Microct and preparation of β-TCP granular material by polyurethane foam method

Commercial ß-tricalcium phosphate (ß-TCP) is commercialy available in granules manufactured by sintering of powders. We have evaluated the different steps of the manufacturing process of ß-TCP ceramics granules prepared from blocks obtained with the polyurethane foam technology. Three types of slurry were prepared with 10, 15 and 25 g of ß-TCP per gram of polyurethane foam. Analysis was done by scanning electron microscopy, EDX, Raman spectroscopy and microcomputed tomography combined with image analysis. A special algorithm was used to identify the internal microporosity (created by the calcination of the foam) from the internal macroporosity due to the spatial repartition of the material. The low ß-TCP dosages readily infiltrated the foam and the slurry was deposited along the polymer rods. On the contrary, the highest concentration produced inhomogeneous infiltrated blocks and foam cavities appeared completely filled in some areas. 2D microcomputed sections and reconstructed 3D models evidenced this phenomenon and the frequency distribution of the thickness and separation of material trabeculae confirmed the heterogeneity of the distribution. When crushed, blocks prepared with the 25 g slurry provided the largest and irregular granulates

Does milling one-piece titanium dental implants induce osteocyte and osteoclast changes?

SummaryOne-piece dental implants avoid adverse effects sometimes associated with the traditional implant-abutment interface and may provide a suitable alternative to two-piece implants; however, one-piece implants often need in situ milling, which may exacerbate cell apoptosis from excessive heat at the bone-implant interface and induce secondary crestal bone loss. Twelve implants were placed in the metaphyses of two sheep under general anesthesia. Six implants were milled with a diamond bur while the other six implants remained intact. Animals were euthanized after four days, and bone blocks were harvested. Bone samples were studied without decalcification. Osteocytes were stained with Hoechst 33342 and osteoclasts by the TRAcP reaction. Both cell types, in the cortical and trabecular bone around the implant\u27s cervical region, were counted utilizing morphometric methods. Values were compared to areas at a distance from the cervical region. No difference was observed between milled and unmilled implants, which suggested that the amount of generated heat did not provoke osteocyte loss or induce osteoclastogenesis. Intraoral abutment preparations did not increase cellular apoptosis at the bone-implant interface after four days in the ovine model

Three-Dimensional Characterization of the Vascular Bed in Bone Metastasis of the Rat by Microcomputed Tomography (MicroCT)

BackgroundAngiogenesis contributes to proliferation and metastatic dissemination of cancer cells. Anatomy of blood vessels in tumors has been characterized with 2D techniques (histology or angiography). They are not fully representative of the trajectories of vessels throughout the tissues and are not adapted to analyze changes occurring inside the bone marrow cavities. Methodology/Principal Findings We have characterized the vasculature of bone metastases in 3D at different times of evolution of the disease. Metastases were induced in the femur of Wistar rats by a local injection of Walker 256/B cells. Microfil®, (a silicone-based polymer) was injected at euthanasia in the aorta 12, 19 and 26 days after injection of tumor cells. Undecalcified bones (containing the radio opaque vascular casts) were analyzed by microCT, and a first 3D model was reconstructed. Bones were then decalcified and reanalyzed by microCT; a second model (comprising only the vessels) was obtained and overimposed on the former, thus providing a clear visualization of vessel trajectories in the invaded metaphysic allowing quantitative evaluation of the vascular volume and vessel diameter. Histological analysis of the marrow was possible on the decalcified specimens. Walker 256/B cells induced a marked osteolysis with cortical perforations. The metaphysis of invaded bones became progressively hypervascular. New vessels replaced the major central medullar artery coming from the diaphyseal shaft. They sprouted from the periosteum and extended into the metastatic area. The newly formed vessels were irregular in diameter, tortuous with a disorganized architecture. A quantitative analysis of vascular volume indicated that neoangiogenesis increased with the development of the tumor with the appearance of vessels with a larger diameter. Conclusion This new method evidenced the tumor angiogenesis in 3D at different development times of the metastasis growth. Bone and the vascular bed can be identified by a double reconstruction and allowed a quantitative evaluation of angiogenesis upon time

Mitochondrial oxodicarboxylate carrier deficiency is associated with mitochondrial DNA depletion and spinal muscular atrophy-like disease.

PURPOSE: To understand the role of the mitochondrial oxodicarboxylate carrier (SLC25A21) in the development of spinal muscular atrophy-like disease. METHODS: We identified a novel pathogenic variant in a patient by whole-exome sequencing. The pathogenicity of the mutation was studied by transport assays, computer modeling, followed by targeted metabolic testing and in vitro studies in human fibroblasts and neurons. RESULTS: The patient carries a homozygous pathogenic variant c.695A>G; p.(Lys232Arg) in the SLC25A21 gene, encoding the mitochondrial oxodicarboxylate carrier, and developed spinal muscular atrophy and mitochondrial myopathy. Transport assays show that the mutation renders SLC25A21 dysfunctional and 2-oxoadipate cannot be imported into the mitochondrial matrix. Computer models of central metabolism predicted that impaired transport of oxodicarboxylate disrupts the pathways of lysine and tryptophan degradation, and causes accumulation of 2-oxoadipate, pipecolic acid, and quinolinic acid, which was confirmed in the patient's urine by targeted metabolomics. Exposure to 2-oxoadipate and quinolinic acid decreased the level of mitochondrial complexes in neuronal cells (SH-SY5Y) and induced apoptosis. CONCLUSION: Mitochondrial oxodicarboxylate carrier deficiency leads to mitochondrial dysfunction and the accumulation of oxoadipate and quinolinic acid, which in turn cause toxicity in spinal motor neurons leading to spinal muscular atrophy-like disease

A Composite Biomaterial: Poly 2 (Hydropoxyethyl) Methacrylate / Alkaline Phosphatase Initiates Mineralization In Vitro

Bone substitutes are nowadays largely used in orthopedic surgery but they lack osteoinductive properties. Poly (2-hydroxyethyl methacrylate) (pHEMA) has numerous biomedical applications. Alkaline phosphatase (AlkP), an ectoenzyme elaborated by osteoblasts, initiates bone mineralization by hydrolyzing organic phosphates before calcium-phosphorus deposition. We have immobilized AlkP in pHEMA in a copolymerization technic. Histochemical study revealed that AlkP has retained its biological activity . Image analysis of sections using a tessellation method showed a lognormal distribution of the area of tessels around AlkP particles thus confirming an homogeneous distribution of the enzyme in the polymer. Pellets of pHEMA and pHEMA + AlkP were incubated with synthetic body fluids containing either inorganic or organic phosphates (ß-glycerophosphate). Mineral deposits with a round shape (calcospherites) were obtained on pHEMA + AlkP pellets incubated in the presence of organic phosphates. No deposits were observed on pHEMA in either incubating conditions or on pHEMA + AlkP incubated with inorganic phosphates. Calcospherites were observed by transmission and scanning electron microscopy. They appeared composed of minute single tablets packed together. X-ray microanalysis showed a Ca/P ratio of 1.42 and X-ray diffraction identified hydroxylapatite. AlkP entrapped in an hydrogel is able to initiate mineralization in vitro by a mechanism that closely mimics the cartilage/bone mineralization in vivo