Introduction et al undifferentiated cells et al s et al @univ-reims.fr Introduction Hydroxyapatite

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Text of Introduction et al undifferentiated cells et al s et al @univ-reims.fr Introduction Hydroxyapatite

  • New electrodeposited Coating Limits Osteoclastogenesis In Vitro Camille Bour1, Aurélie Moniot1, Richard Drevet2, Christine Guillaume1, Dominique Laurent-Maquin1, Sophie C. Gangloff1, Hicham Benhayoune2, Frédéric Velard1

    1EA 4691 BIOS, URCA, France 2EA 4695 LISM, URCA, France

    frederic.velard@univ-reims.fr

    Introduction Hydroxyapatite (HAp) is widely used for filling bone defects or for prosthesis coating. HAp is known for its capability to mount an inflammatory response which if uncontrolled, could lead to implant loss due to osteoclasts (OCs) differentiation and activation (Velard et al, Acta Biomater. 2013). To improve the bioactivity of the implant, electrodeposited calcium-deficient HAp (CaDHAp) was elaborated (Benhayoune H et al, Patent WO2010055231; Drevet et al, J. Med. Sci. Mat. Med. 2011 and RCS Adv. 2013). Previous short term studies have proved that ceramics coatings enhanced cytokine inflammatory response compared to titanium alloy (Ti6Al4V) and more interestingly, CaDHAp coatings were less inflammatory than HAp (Velard et al, ECB 2011). Based on these results we wondered if early cytokine regulation could impact osteoclastogenesis. Here, we have studied human primary monocytes differentiation into OCs in contact with CaDHAp, HAp or Ti6Al4V alone.

    Conclusion and Perspectives Combined with our previous data collected on early monocytic inflammatory response, these results suggest that CaDHAp might be used as a novel coating for

    prosthesis to improve implant integration and lifespan as it controls inflammatory environment and limits osteoclastogenesis, both responsible for aseptic loosening.

    The authors warmly thank Pr P Nguyen and Ms C Macé (EA 3800 HERVI, URCA) for elutriations and PICT (URCA) platform for imagery. This work was supported by région Champagne-Ardenne Emergence Nanosurf and ANR program Nanobonefiller.

    Ti6Al4V Ti6Al4V

    HAp

    2.106 cells/mL ± osteoclastic differentiation environment (M-CSF and RANK-L)

    CaDHAp

    Counterflow elutriated monocytes (> 97% by flow cytometry)

    2 days 21 days

    Ti6Al4V

    60°C Electrolyte: 0.042 M Ca(NO3)2.4H2O 0.025 M NH4(H2PO4) pH 4.4 + 9% H2O2 for stoichiometric HAp

    Ti6Al4V

    Cathode

    Reference electrode

    Counter electrode

    Ultrasonication in acetone and demineralized water

    HNO3 6% HF 3% Ti6Al4V

    ELECTRODEPOSITION (pulsed)

    Cells Morphology: SEM /Phalloidin-AlexaFluor®488 /DAPI staining OCs Count: Phalloidin-AlexaFluor®488 /DAPI staining /TRAP Gelatinolytic activity: Zymography mRNA expression of OCs markers: qPCR (CTSK, CALCR, RANK, MMP-9)

    Preparation of coatings on Ti6Al4V Cells culture Analyses

    Materials and Method

    Results and Discussion

    Osteoclastic markers kinetics of mRNA expression on different materials (n=7). CALCR and RANK mRNA variation was evaluated by RT-PCR analysis using the 2-ΔΔCt method (versus

    internal control RPS18). * means p < 0.05 between considered conditions, £ versus control and & versus Ti6Al4V. Non parametric exact stratified Wilcoxon Mann Whitney test.

    Increased expression of osteoclastic markers over time is not dependent of substrate

    (A) Representative photographs of Phalloidin-AlexaFluor®488/DAPI staining, (B) Scanning Electron Microscopy and (C) TRAP staining

    of undifferentiated or differentiated monocytes after 12 days, n=8. Magnification: (A) x 20, (B) x 1500 and (C) x 10

    M-CSF+RANK-L BASAL A

    B

    C

    Osteoclasts formation requires M-CSF and RANK-L

    mRNA production was evaluated by RT-qPCR analysis (n=7). Data were shown as variation of CTSK, CALCR, RANK and MMP-9 mRNA using the 2-ΔΔCt method (versus internal control RPS18). Red bar represents median value. * means p < 0.05 when compared with non-stimulated cells.

    Non parametric exact stratified Wilcoxon Mann Whitney test.

    Markers of Osteoclastic differentiation expression

    (A) Representative photographs of Phalloidin-AlexaFluor®488/DAPI staining after 21 days of culture without or with M-CSF and RANK-L (Magnification x20). (B) Kinetic of

    OCs differentiation based on cell count per field from Phalloidin-AlexaFluor®488/DAPI staining. n=13. $ means p < 0.05 when compared with previous time point. Non

    parametric exact stratified Wilcoxon Mann Whitney test.

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    Representative photographs (n=2) of Phalloidin-AlexaFluor®488/DAPI staining after 12 days of culture with M-CSF and RANK-L on Ti6Al4V, HAp and CaDHAp

    materials. Arrowheads highlight osteoclastic cells. Magnification x20.

    HAp CaDHAp

    Control Ti6Al4V

    Ceramics coatings reduce osteoclastic formation versus Ti6Al4V substrate

    Effect of the different materials on proMMP-9-related gelatinolytic activity in cell culture supernatants. (A) Representative photographs of zymograms (gelatinolytic activity is

    seen as white bands against dark background). (B) Quantification of gelatinolytic activity by image analysis. (n=7). $ means p < 0.05 when compared with non-stimulated cells.

    * means p < 0.05 when compared with control. Non parametric exact stratified Wilcoxon Mann Whitney test.

    CaDHAp decreases MMP-9 activity

    ProMMP-9 (92 kDa)

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