Introduction et al undifferentiated cells et al s et al · [email protected]...

1
New electrodeposited Coating Limits Osteoclastogenesis In Vitro Camille Bour 1 , Aurélie Moniot 1 , Richard Drevet 2 , Christine Guillaume 1 , Dominique Laurent-Maquin 1 , Sophie C. Gangloff 1 , Hicham Benhayoune 2 , Frédéric Velard 1 1 EA 4691 BIOS, URCA, France 2 EA 4695 LISM, URCA, France [email protected] 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 2.10 6 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(NO 3 ) 2 .4H 2 O 0.025 M NH 4 (H 2 PO 4 ) pH 4.4 + 9% H 2 O 2 for stoichiometric HAp Ti6Al4V Cathode Reference electrode Counter electrode Ultrasonication in acetone and demineralized water HNO 3 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. 0 5 10 15 20 25 30 4 9 14 19 Osteoclasts (% per well) Time (days) $ $ $ $ M-CSF+RANK-L BASAL 5 days of culture were sufficient to observe osteoclasts formation A B 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) A B 0 10000 20000 30000 40000 50000 60000 Total gelatinolytic activity (IOD) * Control Ti6Al4V HAp CaDHAp $ $ $ undifferentiated cells differentiated cells CALCR RANK MMP-9 CTSK * * 0,00001 0,0001 0,001 0,01 0,1 1 10 100 Variation of mRNA expression versus internal reference differentiated cells undifferentiated cells 0,001 0,01 0,1 1 RANK mRNA variation versus internal reference Day 5 Day 12 Day 9 Control Ti6Al4V HAp CaDHAp * * £ £ 0,00001 0,0001 0,001 0,01 0,1 1 CALCR mRNA variation versus internal reference Day 5 Day 12 Day 9 * * * * * * * * Control Ti6Al4V HAp CaDHAp & &

Transcript of Introduction et al undifferentiated cells et al s et al · [email protected]...

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

[email protected]

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.

0

5

10

15

20

25

30

4 9 14 19

Ost

eo

cla

sts

(% p

er

well

)

Time (days)

$

$

$

$

M-CSF+RANK-L BASAL

5 days of culture were sufficient to observe osteoclasts formation

A

B

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)

A

B

0

10000

20000

30000

40000

50000

60000

To

tal

gela

tino

lyti

c a

cti

vity

(IO

D)

*

Control Ti6Al4V HAp CaDHAp

$

$

$

undifferentiated cells

differentiated cells

CALCR RANK MMP-9CTSK

*

*

0,00001

0,0001

0,001

0,01

0,1

1

10

100

Var

iati

on o

f m

RN

A e

xpre

ssio

n v

ers

us

inte

rnal

refe

rence

differentiated cells

undifferentiated cells

0,001

0,01

0,1

1

RA

NK

mR

NA

var

iati

on v

ers

us

inte

rnal

re

fere

nce

Day 5

Day 12

Day 9

Control Ti6Al4V HAp CaDHAp

**

£

£

0,00001

0,0001

0,001

0,01

0,1

1

CA

LC

R m

RN

A v

aria

tio

n v

ers

us

inte

rnal

re

fere

nce

Day 5

Day 12

Day 9

*

**

**

**

*

Control Ti6Al4V HAp CaDHAp

&

&