Stéphane Durual

APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa

The bone marrow constitutes a favorable environment for long-lived antibody-secreting plasma cells, providing blood-circulating antibody. Plasma cells are also present in mucosa-associated lymphoid tissue (MALT) to mediate local frontline immunity, but how plasma cell survival there is regulated is not known. Here we report that a proliferation-inducing ligand (APRIL) promoted survival of human upper and lower MALT plasma cells by upregulating expression of the antiapoptotic proteins bcl-2, bcl-xL, and mcl-1. The in situ localization of APRIL was consistent with such a prosurvival role in MALT. In upper MALT, tonsillar epithelium produced APRIL. Upon infection, APRIL production increased considerably when APRIL-secreting neutrophils recruited from the blood infiltrated the crypt epithelium. Heparan sulfate proteoglycans (HSPGs) retained secreted APRIL in the subepithelium of the infected zone to create APRIL-rich niches, wherein IgG-producing plasma cells accumulated. In lower MALT, neutrophils were the unique source of APRIL, giving rise to similar niches for IgA-producing plasmocytes in villi of lamina propria. Furthermore, we found that mucosal humoral immunity in APRIL-deficient mice is less persistent than in WT mice. Hence, production of APRIL by inflammation-recruited neutrophils may create plasma cell niches in MALT to sustain a local antibody production.

Growth differentiation factor 15 production is necessary for normal erythroid differentiation and is increased in refractory anaemia with ring-sideroblasts

The disturbed erythropoiesis in patients with refractory anaemia with ring‐sideroblasts (RARS) is characterized by intramedullary apoptosis of erythroid precursors and increased iron accumulation in mitochondria. To gain insight into these pathophysiological mechanisms we compared the gene expression profile (GEP) of erythroid precursors from RARS patients to the GEP of normal erythroid precursors. Three hundred sixty four probe sets were up‐, and 253 probe sets downregulated in RARS cells. Interestingly, Growth Differentiation factor 15 (GDF15), a cytokine from the TGFβ family, was dramatically upregulated in all RARS patients. Measurement of GDF15 in the sera from twenty RARS patients confirmed this finding by showing significantly, 7·2‐fold, increased protein levels (3254 ± 1400 ng/ml vs. 451 ± 87 ng/ml in normals). In vitro studies demonstrated erythroid‐specific production of GDF15 and dependence on erythropoietin. Induction of apoptosis by arsenic trioxide, a drug which acts via reduction of the mitochondrial membrane potential, also stimulated GDF15 production. Downregulation of endogenous GDF15 production in erythoblasts by specific siRNA led to diminished erythroid differentiation. Taken together, our findings demonstrate a new role for GDF15 in normal erythropoiesis as well as in the ineffective erythropoiesis of RARS patients.

Lentiviral PU.1 overexpression restores differentiation in myeloid leukemic blasts

PU.1, a transcription factor of the ETS family, plays a pivotal role in normal hematopoiesis, and particularly in myeloid differentiation. Altered PU.1 function is possibly implicated in leukemogenesis, as PU.1 gene mutations were identified in some patients with acute myeloid leukemia (AML) and as several oncogenic products (AML1-ETO, promyelocytic leukemia-retinoic acid receptor alpha, FMS-like receptor tyrosine kinase 3 internal tandem duplication) are associated with PU.1 downregulation. To demonstrate directly a role of PU.1 in the blocked differentiation of leukemic blasts, we transduced cells from myeloid cell lines and primary blasts from AML patients with a lentivector encoding PU.1. In NB4 cells we obtained increases in PU.1 mRNA and protein, comparable to increases obtained with all-trans retinoic acid-stimulation. Transduced cells showed increased myelomonocytic surface antigen expression, decreased proliferation rates and increased apoptosis. Similar results were obtained in primary AML blasts from 12 patients. These phenotypic changes are characteristic of restored blast differentiation. PU.1 should therefore constitute an interesting target for therapeutic intervention in AML.

Hydrothermal degradation of a 3Y-TZP translucent dental ceramic: A comparison of numerical predictions with experimental data after 2 years of aging

OBJECTIVES The purpose of the study was to assess the hydrothermal resistance of a translucent zirconia with two clinical relevant surface textures by means of accelerated tests (LTD) and to compare predicted monoclinic fractions with experimental values measured after two years aging at 37°C. METHODS Polished (P) and ground (G) specimens were subjected to hydrothermal degradation by exposure to water steam at different temperatures and pressures. The t-m phase transformation was quantified by grazing incidence X-ray diffraction (GIXDR). The elastic modulus and hardness before- and after LTD were determined by nanoindentation. RESULTS G specimens presented a better resistance to hydrothermal degradation than P samples. Activation energies of 89 and 98kJ/mol and b coefficients of 2.0×10(-5) and 1.8×10(-6) were calculated for P and G samples respectively. The coefficients were subsequently used to predict transformed monoclinic fractions at 37°C. A good correlation was found between the predicted values and the experimental data obtained after aging at 37°C during 2 years. Hydrothermal degradation led to a significant decrease of the elastic moduli and hardness in both groups. SIGNIFICANCE The dependency of the t-m phase transformation rate on temperature must be determined to accurately predict the hydrothermal behavior of the zirconia ceramics at oral temperatures. The current prevailing assumption, that 5h aging at 134°C corresponds to 15-20 years at 37°C, will underestimate the transformed fraction of the translucent ceramic at 37°C. In this case, the mechanical surface treatment influences the ceramics transformability. While mild grinding could potentially retard the hydrothermal transformation, polishing after occlusal adjustment is recommended to prevent wear of the antagonist teeth and maintain structural strength.

Effect of different surface treatments on the hydrothermal degradation of a 3Y-TZP ceramic for dental implants

OBJECTIVES Implant surface modifications are intended to enhance bone integration. The objective of this study was to assess the effect of different surface treatments on the resistance to hydrothermal degradation, hardness and elastic modulus of a 3Y-TZP ceramic used for dental implants. METHODS Samples grouped according to their surface morphologies (AS, as-sintered; C, coated; P, dry-polished; R, roughened; PA, polished and annealed; RA, roughened and annealed) were subjected to accelerated hydrothermal degradation (LTD) by exposure to water steam (134°C, 2bars) for 100h. The t-m phase transformation was quantified by grazing incidence X-ray diffraction (GIXDR) and by combined focused ion beam and scanning electron microscopy (FIB-SEM). Elastic modulus and hardness before- and after prolonged aging (100h) were assessed by nanoindentation. RESULTS AS and C specimens presented a better resistance to hydrothermal degradation than P and R samples. After prolonged aging, the depth of the monoclinic transformed layer ranged from 11μm to 14μm. Hydrothermal degradation led to a significant decrease of elastic modulus and hardness. SIGNIFICANCE Surface treatments affected the resistance to hydrothermal degradation of the 3Y-TZP ceramic. Dry mechanical surface modifications should be avoided since a high t-m transformation rate associated to the initial monoclinic content was observed. Annealing was useful to reverse the initial t-m transformation, but did not improve the resistance to hydrothermal degradation.

TiNOx coatings on roughened titanium and CoCr alloy accelerate early osseointegration of dental implants in minipigs

INTRODUCTION Titanium nitride oxide (TiNOx) coatings are known for their biocompatibility, hardness and high resistance to corrosion and wear. Further, they can be applied by plasma vapor deposition onto a wide variety of metallic, mineral, or organic substrates. In cell cultures, TiNOx coatings applied onto SLA (sandblasted, large grit, acid etched)-roughened titanium surfaces increased human primary osteoblast proliferation by 1.5 times in the first 2 weeks after seeding, while maintaining a high degree of cell differentiation. Therefore, the objectives of the present study were (i) to determine whether these findings would translate into the enhanced osseointegration of TiNOx-coated implants in vivo and (ii) to compare the osseointegration of Ti-SLA (titanium-SLA) and CoCr-SLA (cobalt-chromium-SLA) implants coated with TiNOx. METHODS Forty-eight cylinders made of Ti-SLA, Ti-SLA-TiNOx (TiNOx-coated Ti-SLA) and CoCr-SLA-TiNOx (TiNOx-coated CoCr-SLA) were implanted into the lower jawbone of 8 minipigs. The bone-to-implant contact was determined after 1 week, 2 weeks, 1 month and 3 months. RESULTS Osseointegration proceeded normally on all 3 surfaces, with equal activity after the first week of healing. After 2 weeks, bone-to-implant contact was 1.8 times higher on TiNOx coatings, either deposited on Ti or on CoCr. These differences fell off after 1 and 3 months of healing. CONCLUSIONS When compared to standard SLA titanium, TiNOx coatings enhance implant osseointegration during the first month of healing. Furthermore, this stimulating effect is independent of the substrate, leading to similar results whether the coating is applied onto SLA-Ti or onto SLA-CoCr.

Grinding damage assessment for CAD-CAM restorative materials

OBJECTIVES To assess surface/subsurface damage after grinding with diamond discs on five CAD-CAM restorative materials and to estimate potential losses in strength based on crack size measurements of the generated damage. METHODS The materials tested were: Lithium disilicate (LIT) glass-ceramic (e.max CAD), leucite glass-ceramic (LEU) (Empress CAD), feldspar ceramic (VM2) (Vita Mark II), feldspar ceramic-resin infiltrated (EN) (Enamic) and a composite reinforced with nano ceramics (LU) (Lava Ultimate). Specimens were cut from CAD-CAM blocs and pair-wise mirror polished for the bonded interface technique. Top surfaces were ground with diamond discs of respectively 75, 54 and 18μm. Chip damage was measured on the bonded interface using SEM. Fracture mechanics relationships were used to estimate fracture stresses based on average and maximum chip depths assuming these to represent strength limiting flaws subjected to tension and to calculate potential losses in strength compared to manufacturers data. RESULTS Grinding with a 75μm diamond disc induced on a bonded interface critical chips averaging 100μm with a potential strength loss estimated between 33% and 54% for all three glass-ceramics (LIT, LEU, VM2). The softer materials EN and LU were little damage susceptible with chips averaging respectively 26μm and 17μm with no loss in strength. Grinding with 18μm diamond discs was still quite detrimental for LIT with average chip sizes of 43μm and a potential strength loss of 42%. SIGNIFICANCE It is essential to understand that when grinding glass-ceramics or feldspar ceramics with diamond discs surface and subsurface damage are induced which have the potential of lowering the strength of the ceramic. Careful polishing steps should be carried out after grinding especially when dealing with glass-ceramics.

Grinding damage assessment on four high-strength ceramics

OBJECTIVES The purpose of this study was to assess surface and subsurface damage on 4 CAD-CAM high-strength ceramics after grinding with diamond disks of 75 μm, 54 μm and 18 μm and to estimate strength losses based on damage crack sizes. METHODS The materials tested were: 3Y-TZP (Lava), dense Al2O3 (In-Ceram AL), alumina glass-infiltrated (In-Ceram ALUMINA) and alumina-zirconia glass-infiltrated (In-Ceram ZIRCONIA). Rectangular specimens with 2 mirror polished orthogonal sides were bonded pairwise together prior to degrading the top polished surface with diamond disks of either 75 μm, 54 μm or 18 μm. The induced chip damage was evaluated on the bonded interface using SEM for chip depth measurements. Fracture mechanics were used to estimate fracture stresses based on average and maximum chip depths considering these as critical flaws subjected to tension and to calculate possible losses in strength compared to manufacturers data. RESULTS 3Y-TZP was hardly affected by grinding chip damage viewed on the bonded interface. Average chip depths were of 12.7±5.2 μm when grinding with 75 μm diamond inducing an estimated loss of 12% in strength compared to manufacturers reported flexural strength values of 1100 MPa. Dense alumina showed elongated chip cracks and was suffering damage of an average chip depth of 48.2±16.3 μm after 75 μm grinding, representing an estimated loss in strength of 49%. Grinding with 54 μm was creating chips of 32.2±9.1 μm in average, representing a loss in strength of 23%. Alumina glass-infiltrated ceramic was exposed to chipping after 75 μm (mean chip size=62.4±19.3 μm) and 54 μm grinding (mean chip size=42.8±16.6 μm), with respectively 38% and 25% estimated loss in strength. Alumina-zirconia glass-infiltrated ceramic was mainly affected by 75 μm grinding damage with a chip average size of 56.8±15.1 μm, representing an estimated loss in strength of 34%. All four ceramics were not exposed to critical chipping at 18 μm diamond grinding. CONCLUSIONS Reshaping a ceramic framework post sintering should be avoided with final diamond grits of 75 μm as a general rule. For alumina and the glass-infiltrated alumina, using a 54 μm diamond still induces chip damage which may affect strength. Removal of such damage from a reshaped framework is mandatory by using sequentially finer diamonds prior to the application of veneering ceramics especially in critical areas such as margins, connectors and inner surfaces.

Medium-Term Function of a 3D Printed TCP/HA Structure as a New Osteoconductive Scaffold for Vertical Bone Augmentation: A Simulation by BMP-2 Activation

Introduction: A 3D-printed construct made of orthogonally layered strands of tricalcium phosphate (TCP) and hydroxyapatite has recently become available. The material provides excellent osteoconductivity. We simulated a medium-term experiment in a sheep calvarial model by priming the blocks with BMP-2. Vertical bone growth/maturation and material resorption were evaluated. Materials and methods: Titanium hemispherical caps were filled with either bare- or BMP-2 primed constructs and placed onto the calvaria of adult sheep (n = 8). Histomorphometry was performed after 8 and 16 weeks. Results: After 8 weeks, relative to bare constructs, BMP-2 stimulation led to a two-fold increase in bone volume (Bare: 22% ± 2.1%; BMP-2 primed: 50% ± 3%) and a 3-fold decrease in substitute volume (Bare: 47% ± 5%; BMP-2 primed: 18% ± 2%). These rates were still observed at 16 weeks. The new bone grew and matured to a haversian-like structure while the substitute material resorbed via cell- and chemical-mediation. Conclusion: By priming the 3D construct with BMP-2, bone metabolism was physiologically accelerated, that is, enhancing vertical bone growth and maturation as well as material bioresorption. The scaffolding function of the block was maintained, leaving time for the bone to grow and mature to a haversian-like structure. In parallel, the material resorbed via cell-mediated and chemical processes. These promising results must be confirmed in clinical tests.

TiNOx coatings increase human primary osteoblasts proliferation independently of the substrate: a short report.

INTRODUCTION When applied onto SLA titanium, Titanium Nitride Oxide (TiNOx) coatings are known for their stimulating effect on osteoblast proliferation while maintaining a high degree of differentiation. OBJECTIVE To determine whether the stimulating effect of TiNOx coatings is or is not dependent of the metal substrate. METHODS A TiNOx coating was deposited on roughened stainless steel (SS-SLA-TiNOx). The proliferation and differentiation of human primary osteoblasts were determined and compared to that observed on standard SLA titanium. To this end, cell proliferation and gene expression were monitored for a time course of 3 weeks, with or without additional stimulation by 100 nM 1.25(OH)2 vitamin D3. RESULTS A 1.5 fold increase in the proliferation rate of cells grown on SS-SLA-TiNOx as compared to Ti-SLA surfaces was observed. A high level of differentiation was also noted. CONCLUSION TiNOx coatings yield similar proliferation and differentiation rates when applied onto roughened Ti and SS. This might translate into a more effective osseointegration of endosseous implants made of stronger metals than Ti.