Effect of gypsum on proliferation and differentiation of MC3T3-E1 mouse osteoblastic cells

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Effect of gypsum on proliferation and differentiation of MC3T3-E1 mouse

osteoblastic cells

Á. Lazáry, B. Balla, J.P. Kósa, K. Bácsi, Z. Nagy, I. Takács, P.P. Vargaa, G. Speer, P.

Lakatos

1st Department of Medicine, Semmelweis University, Korányi S. u. 2/a, Budapest, H-1083

Hungary,

a Center of Spinal Disorders, Buda Health Center, Királyhágó u. 1-3., Budapest, H-1126

Hungary

Keywords: calcium sulfate, bone graft, gene expression, osteoinduction, bone healing

Corresponding author: Áron Lazáry MD, 1st Department of Medicine, Semmelweis

University, Korányi S. u. 2/a, Budapest, H-1083 Hungary. Tel.: +36 1 2100278/1566; Fax:

+36 1 2104874; E-mail address: [email protected]

Running title: Effect of gypsum on osteoblasts

* Title Page

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Abstract

Recently, calcium sulfate dihydrate has been demonstrated as safe biodegradable

osteoconductive bone void filler. However, its exact mechanism of action on bone cells is yet

unknown. In this study, the influence of gypsum on gene expression and proliferation of

MC3T3-E1 mouse pre-osteoblastic cells was investigated. Cells were cultured on gypsum

disc, slice, polymethylmethacrylate, or plastic culture plate for 15 days. Cell viability, alkaline

phosphatase (ALP) activity and expression profile of 15 genes involved in bone metabolism

were measured in cultures. Cell proliferation on gypsum was increased by almost twofold,

while an inhibitory effect of polymethylmethacrylate on proliferation rate of osteoblasts was

noted. Cells cultured on gypsum disc surface exhibited an increased ALP activity and

markedly different gene expression profile. Quantitative real-time PCR data indicated the

expression of genes that might provide a basis for an osteoinductive potential. MC3T3-E1

cells expressed genes typical of bone fracture healing like type II collagen and fibronectin 1.

These effects might be related to the calcium content of gypsum and mediated likely via

SMAD3. Our results suggest that gypsum can support new bone formation by its calcium

content and modulatory effect on gene expression profile of bone cells.

* Abstract

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Introduction

The use of calcium sulfate as bone void filler has a long clinical history. In the middle

of the last century, Peltier at al. [1] collected extensive data supporting the efficiency and

safety of Plaster of Paris (calcium sulfate hemihydrate). A multicenter trial was published by

Kelly at al. [2] in 2001 where authors have used Osteoset® (surgical grade calcium sulfate,

Wright Medical Technology, Arlington TN) to fill bone defects caused by benign bone

tumors, trauma, cyst etc. altogether in 109 cases. They concluded that surgical grade calcium

sulfate is reliable, convenient, safe and readily available bone graft substitute that yields

consistent results. Calcium sulfate has been recently introduced in new fields of indications.

Borrelli at al. [3] have successfully treated 26 patients who had an operation of a nonunion

osseous defect caused by trauma with a mixture of autogenous iliac bone and Osteoset®. In

2005, Chen at al. [4] published the use of calcium sulfate in posterolateral spine fusion. Their

conclusion was that surgeons could use calcium sulfate combined with locally harvested

morselized bone as an artificial bone expander with a good fusion rate.

Gypsum – the dihydrate form of calcium sulfate (CaSO4 * 2 H2O) – can be found in

nature, where the mineral is one perfect crystal, in contrast to gypsum made from Plaster of

Paris – hemihydrate form of calcium sulfate (CaSO4 * ½ H2O) – where a lot of small crystals

develop next to each other. Gypsum dissolves weakly in water to Ca2+ and SO42-.

The extracellular calcium concentration in sites of bone remodeling has been measured

as high as 40 mM [5]. Moderate high extracellular Ca2+ is a chemotactic and proliferating

signal for osteoblasts and stimulate the differentiation of MC3T3-E1 pre-osteoblasts [6-8],

suggesting that extracellular calcium plays an important role in the regulation of bone cells.

A number of positive clinical experiences are available with calcium sulfate in bone

substitution procedures, however, the exact mechanism of action of calcium sulfate is poorly

understood. The objective of the present study was to examine the Ca2+ concentration above

* Manuscript

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gypsum surfaces, as well as to demonstrate the importance of crystal structure of gypsum in

bone substitution and to study the molecular effects of gypsum on osteoblasts compared with

other (polymethylmethacrylate - PMMA) bone substitute materials generally used in clinical

practice.

Materials and Methods

Cell cultures

MC3T3-E1 mouse pre-osteoblast cells were used for cell cultures. Cells were fed

twice a week with α-MEM (Sigma-Aldrich Inc., St. Louis, MO, USA) containing 10% fetal

calf serum and antibiotics (Sigma-Aldrich Inc., St. Louis, MO, USA). There was a standard

Ca2+ level of 1.8 mM and a concentration of ascorbic acid of 25 g/ml in the culture medium.

Cultures were grown in 5% CO2 at 37°C and 85% humidity.

Culture surfaces

Cells were plated on gypsum disc, mineral gypsum slice, PMMA and plastic culture

plate (CP). Gypsum disc was prepared by suspending heat-sterilized CaSO4* ½ H2O (Sigma-

Aldrich Inc., St. Louis, MO, USA) in distilled water in 50% weight concentration. Mineral

gypsum slices were cut from a gypsum crystal mined in Hungary. These slices were sterilized

in a formaldehyde sterilizer. PMMA surface was made by extra low viscosity bone cement

(Cemex® XL – Tecres S.p.A., Verona, Italy).

Detection of Ca2+ concentration

Ca2+ level was measured from culture media to determine the base Ca2+ concentration

above different forms of gypsum, such as gypsum discs and mineralized gypsum slices on 3,

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6, 10, 14 and 21 days. In order to measure it, 0.5 ml of culture medium was diluted with α-

MEM to 5 ml and an Olympus AU2700 analyzer (Olympus Life and Material Science Europa

GmbH, Hamburg, Germany) was used to determine total Ca2+ concentration based on reaction

between Ca2+ and o-cresolphthalein.

Proliferation assay

MC3T3-E1 osteoblasts were plated at 10.400 cells per cm2 surface area on different

forms of gypsum, PMMA and CP. In case of some CP cultures, 150 μl of 0.5 M CaCl2 was

added to 3 ml medium to achieve a final Ca2+ concentration of 25.5 mM. We used CellTiter-

Glo® Luminescent Cell Viability Assay (Promega Co., Madison, WI, USA) to determining

the number of viable cells in cultures. At 4h, and 28h after plating, plates were centrifuged for

10 min at 1000 rpm to bring cells floating in the media to the bottom of the plate and medium

was discarded. A mixture 250 μl CellTiter-Glo® reagent and 250 μl α-MEM was added in

each cultures. Plates were shaken for 3 minutes and incubated at room temperature for 10

minutes. Luminescence intensity was directly proportional to ATP amount and number of

viable cells.

Determination of alkaline phosphatase (ALP) activity

Cell cultures were supplemented with osteogenic medium containing 10 mM β-

glycerol-phosphate (Sigma-Aldrich Inc., St. Louis, MO, USA) and 25 μg/ml ascorbic acid

(Sigma-Aldrich Inc., St. Louis, MO, USA) to ALP study. ALP activity was measured from

the culture media after 15 days of culture with p-nitrophenil-phosphate reaction using an

Olympus AU2700 analyzer as it is described in users manual. ALP activity was normalized to

protein quantity of samples measured by NanoDrop® ND-1000 spectrophotometer

(NanoDrop Technologies, Wilmington, DE, USA) at 280 nm after digesting of attached cells

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using CellLyticTM cell lysis reagent (Sigma-Aldrich Inc., St. Louis, MO, USA).

Gene expression study

MC3T3-E1 cells were plated with non-supplemented α-MEM and with medium

supplemented with 25 mM CaCl2 on plastic culture plate, on gypsum disc, as well as on

PMMA to determine the effect of Ca2+ concentration of 25.5 mM and the effect of gypsum

surface on gene expression profile. Total RNA was isolated from cell cultures of 15 days

using High Pure RNA Isolation Kit (Roche Diagnostics GmbH, Manheim, Germany). RNA

quantity and quality were controlled by NanoDrop® ND-1000 spectrophotometer at 260 nm.

Ten μl of total RNA (50 ng/μl) was incubated with 600 ng random hexamers for 8 min at

65°C. 200 U M-MLV reverse transcriptase, 100 nmol dNTP, 20 U RNazin and 4 μl m-MLV

5x buffer were added to reaction mixture (all reagents of reverse transcription were

purchased form Promega Co., Madison, WI, USA). Final volume of 20 μl of cDNA was

synthesized at 37°C under 60 min. cDNA was amplified by real-time PCR using TaqMan®

Gene Expression Assays (Applied Biosystem, Foster City, CA, USA). Reaction volume was

20 μl containing 1 μl cDNA, 10 μl TaqMan® 2x Universal PCR Master Mix NoAmpErase

UNG, 1 μl predesigned and validated gene-specific TaqMan® Gene Expression Assay 20x

and 8 μl water. ABI Prism 7500 real-time PCR system (Applied Biosystem, Foster City, CA,

USA) was used to amplify the 15 selected genes (Table 1.) from each sample in three parallel

runs on a 96-well optical reaction plate (Applied Biosystem, Foster City, CA, USA) with the

following protocol: 10 minutes denaturizing at 95 °C, and 50 cycles of 15 sec denaturizing at

95 °C, 1 min annealing and extension at 60 °C. Housekeeping gene of GAPDH was used as

internal control in reactions. Relative quantification studies were made from collected data

with 7500 System SDS software 1.3.

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Statistics

Final data are presented as the means of at least three independent measurements. Test

data were analyzed using the SPSS for Windows, release 13.0.1 (SPSS Inc., Chicago, IL,

USA). Results are expressed as mean ± standard error of mean (S.E.M.). Statistical analysis

was performed using unpaired Student’s t-test with a p value of 0.01 or less considered

significant.

Results

Ca2+ concentration in culture medium above gypsum

The mean value of Ca2+ concentration above gypsum discs and mineral gypsum slices

with and without cells was 25.48 ± 0.83 mM after 3 days of culture, and there was no

significant difference among different experimental setups.

Cell shape

Shape of MC3T3-E1 cells was different depending on various culture conditions as

shown on Figure 1. Cells were rather spindle-shaped on gypsum discs (A, B) compared to

culture plates where they were more cubical (F). MC3T3-E1 osteoblasts could not adhere to

mineral gypsum slices (C). This spindle-shaped morphological change could also be noted on

culture plates when medium was supplemented with Ca2+ to a final Ca2+ concentration of 25.5

mM (E). Degenerated and necrotic cells appeared in a high ratio on PMMA surface (D).

Cell viability assay

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Osteoblasts were viable on gypsum discs and they were able to proliferate on it with

an increased ratio compared to PMMA where cells were inhibited to grow. Growth rate of

MC3T3-E1 cells on gypsum discs were similar to plastic culture plates using medium

supplemented with 25 mM of CaCl2 (Figure 2.).

Alkaline phosphatase activity on gypsum

After 15 days of culture, alkaline phosphatase activity measured from supernatants

was significantly higher on gypsum discs and culture plates with Ca2+-supplemented medium

than on culture plates with normal Ca2+ and PMMA. SMAD3 expression in the different cell

cultures has changed with the same tendency (Figure 3.).

Effect of gypsum surface and high Ca2+ level on gene expression profile of MC3T3-E1

osteoblasts

Gene expression data were determined from cultures of 15 days and were normalized

to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the same

culture. Relative expression of studied genes in different cultures is shown on Figure 4.

A different gene expression profile was observed with quantitative real-time PCR on

gypsum compared to culture plate with standard Ca2+. Relative expression of type II collagen

(COL2A1) was more than 130-fold higher (p<0.001) on gypsum surface. Expression of

fibronectin 1 (FN1), SMAD3 and SMAD6 have also significantly increased in cells cultured

on gypsum. Gene expression of type I collagen (COL1A1) was 12-fold increased (p<0.001)

on culture plate with standard Ca2+. Amount of gene specific mRNA of decorin (DCN) and

bone morphogenic protein 4 (BMP4) were decreased on gypsum. Bone sialoprotein (BSP),

osteocalcin (BGLAP) and calcium sensor (CASR) was not expressed in detectable amount on

gypsum disc.

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When Ca2+ concentration was increased in the standard medium to the extent of that

present in the medium above gypsum discs, an 80-fold increase in COL2A1 and a 20-fold

decrease in COL1A1 expression was seen in cultures with high Ca2+ level. SMAD6 and

SMAD3 expressions also significantly increased due to Ca2+ supplementation, similarly to

that seen in gypsum disc cultures. Amount of mRNA of DCN, BMP4 and BSP was decreased

in the presence of high Ca2+ level. CASR and BGLAP have been expressed in cells cultured

in standard α-MEM containing 1.8 mM Ca2+ but were not detectable in cultures with 25.5

mM extracellular Ca2+. The expression profile in osteoblasts cultured in high Ca2+ medium

was similar to that of cells grown above gypsum disc.

MC3T3-E1 cells on gypsum disc expressed a large amount of COL2A1 compared to

PMMA (51-fold difference, p<0.001), while expression of COL1A1 was 7-fold higher on

PMMA. FN1, SMAD6 and SMAD3 were also overexpressed on gypsum disc but expression

of BMP4 and DCN were significantly lower than on PMMA. CASR, BGLAP and BSP were

not expressed on gypsum disc while detectable amount of gene specific mRNA of these genes

was measured in cultures on PMMA. The expression profile in osteoblasts cultured on

PMMA was similar to that of cells grown in culture plates with standard calcium

concentration.

Discussion

In our study, we found that osteoblastic cells can proliferate on gypsum disc with a

significantly higher rate than on PMMA. Physical structure of gypsum appears to be

important for the proper adherence of osteoblasts to surface. Cells cannot attach to mineral

gypsum slices with perfect crystal structure that is not accessible for osteoblasts. However,

MC3T3-E1 cells can adhere to gypsum disc surface when it has been developed from Plaster

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of Paris (CaSO4-hemihydrate) by adding water. In this form of gypsum, a number of smaller

crystals are located side by side ensuring a large molecular surface that can be utilized by

cells for proliferation and matrix production. Cells are more spindle-shaped on gypsum likely

due to the large Ca2+ content of CaSO4-dihdyrate.

ALP activity is a marker of osteoblastic activity, i.e. bone turnover and bone

remodeling. Its level in serum increases during bone healing after fracture [9,10]. Bone cells

adhered to gypsum surface can be stained for alkaline phosphatase [11]. Winn et al. [12] have

reported a decrease in the proliferation ratio and ALP activity of osteoblasts cultured on

surgical grade calcium sulfate pellets. However, they found that ALP activity of these cultures

depends on culture conditions. ALP activity and mineralization of MC3T3-E1 pre-osteoblast

cells are under the influence of extracellular Ca2+ [7,8], and it is likely to be related to the

expression of SMAD3 gene (a critical component of TGFβ signaling pathway) [13]. We have

found an increase in ALP activity in the presence of high Ca2+ in culture medium. In cultures

on gypsum disc and culture plate with Ca2+ supplementation, SMAD3 expression was also

higher than in cultures with standard Ca2+ concentration or on PMMA. This result suggests

that stimulatory effect of high extracellular Ca2+ on ALP activity is mediated, at least partly,

via SMAD3, a transcription factor known to participate in bone formation and healing [14].

In the presence of highly expressed SMAD3, transcription of late markers of

osteogenic differentiation, such as BGLAP and BSP, were decreased or undetectable. These

findings support the results of Sowa at al. [13] that SMAD3 stimulates ALP activity and

mineralization but inhibits osteocalcin expression in MC3T3-E1 cells. They also described

that TGF treatment or SMAD3 transfection affected the shape of osteoblastic cells. As we

have also found, cells are rather spindle shaped in cases of high SMAD3 expression. Changes

in the expression of SMAD6 shown in our experiments also corroborate the important role of

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TGFβ-signaling pathway in osteoblast differentiation. SMAD6 has been demonstrated to have

a role in the negative feedback loop of this process [15].

Our in-vitro results indicated that MC3T3-E1 pre-osteoblast cells differentiate in

distinct ways in different culture conditions used in this study. Gene expression of

extracellular matrix components has markedly changed depending on the different culture

surfaces. The expression profile above gypsum disc has changed to reflect repair processes in

bone. Probably high extracellular Ca2+ level has initiated these effects on different types of

collagen and other small matrix component gene expression since these changes could partly

be reproduced on plastic plates filled with high Ca2+ medium. Recent studies have described

that during bone repair processes an overexpression of COL2A1 and FN1 can be detected

[16,17], and newly formed healing bone can be characterized by the presence of COL2A1

expressed in active osteoblasts and not in chondrocytes [18]. FN1 can play a role in the

scarless wound healing of fractured bone [16]. DCN – like other proteoglycans in bone –

participates in collagen assembly and function but it also acts in embryogenesis and bone cell

differentiation [19]. The gene expression pattern observed above gypsum was identical in our

experiments.

Many authors have supposed that calcium sulfate dihydrate is a safe biodegradable

bone void filler [20,21]; its slow absorption give possibility to fibrovascular tissue ingrowth,

neovascularization [22], and new bone formation [23] in the defect. Walsh et al. [24] have

suggested the idea of an osteoinductive effect elicited by calcium sulfate that was based on

their finding of a high amount of TGF in bone void filled with calcium sulfate pellets. This

effect could be related to the local acidity caused by the dissolution of the pellets.

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Conclusion

Cells plated on gypsum disc express genes important in new bone formation with

different expression ratio compared to polymethylmethacrylate, generally used as a bone void

filler, suggesting that gypsum provides a more efficient environment for bone repair. Thus,

our results underline the former suggestion that gypsum is not only a “passive”

osteoconductive material but it might also has a potential to ostoinductivity due to its special

crystal structure and high calcium content, however further investigations – implantation

experiment in non-osseous tissues e.g. – are required to confirm it.

Acknowledgement

This work was supported by grants from NKFP-1A/002/2004 and NKFP-1A/007/2004.

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Table 1. List of selected genes for gene expression study

Figure 1. Cell shape of MC3T3-E1 cells on different culture surfaces (cultures of 3 days,

standard hematoxilyn staining)

A. gypsum disc, 100x; B. gypsum disc, 600x; C. mineral gypsum slice, 40x; D.

polymethylmethacrylate (PMMA), 100x; E. culture plate with Ca2+ concentration of 25.5

mM, 100x; F. culture plate with Ca2+ level of 1.8 mM, 100x.

Figure 2. Changes of cell number in a 24 h incubation period on different surfaces

Proliferation ratio (PR) on gypsum disc (GYPS) /PR: 1.89 ± 0.091/ was significant higher

than on polymethylmetacrylate bone cement (PMMA) /PR: 0.914 ± 0.052/ and it is not

different to PR on culture plate when medium was supplemented with 25 mM Ca2+ (CP25)

/PR: 2.07 ± 0.060/ (p>0.05). PR was 2.38 ± 0.063 on culture plate without Ca2+

supplementation (data not shown). (*:p<0.01, **:p<0.001)

Figure 3. A. Alkaline phosphatase activity in culture medium above different culture surfaces

ALP activity on gypsum disc (GYPS) was 23.14 ± 4.55 U/g protein, and it was significantly

higher than ALP activity on culture plate (CP), but it was not significantly different from

ALP activity on culture plates when medium was supplemented with 25 mM Ca2+ (CP25)

/ALP: 30.07+-3.04/ (p>0.05). A significant 6-fold increase of ALP activity was detected

between cultures on gypsum disc and cell cultures on polymethylmetacrylate (PMMA) /ALP:

4.08 ± 0.62 U/g protein/. This difference was also noted comparing cells on culture plate with

Ca2+-supplemented medium to cells on culture plate without Ca2+ supplementation (CP)

/ALP: 4.44±0.52/. (*:p<0.01, **:p<0.001)

B. Relative gene expression of SMAD3 on different culture surfaces after 15 days

Captions

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SMAD3 expression was 2.2-fold higher on gypsum disc (GYPS) surface than on

polymethylmetacrylate (PMMA) and 2.6-fold higher than on culture plate (CP). On culture

plates, in case of 25 mM Ca2+ supplementation (CP25) SMAD3 expression increased 3-fold.

(*:p<0.01, **:p<0.001)

Figure 4. Changes of gene expression due to high extracellular Ca2+ and gypsum (see also

Figure 3/b.)

Cells were cultured on culture plate with standard -MEM (CP), on culture plate with -

MEM containing 25.5 mM Ca2+ (CP25), on gypsum disc (GYPS) and on

polymethylmetacrylate (PMMA) for 15 days. Relative expression of selected genes

normalized to GAPDH of each sample are shown in cases where increase or decrease was

1.75-fold or more, and significant (*: p<0.01, **:p<0.001, †: studied gene was not expressed

in detectable amount in this culture)

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Table 1.

Assay IDGene

SymbolGene Name General function

Mm00455918_m1 BGN biglycanExtracellular matrix structural

protein

Mm00432087_m1 BMP4bone morphogenetic

protein 4

Signaling molecule / TGF-beta

superfamily

Mm00432109_m1 BMP8abone morphogenetic

protein 8a

Signaling molecule / TGF-beta

superfamily

Mm00801666_g1 COL1A1procollagen, type I,

alpha 1

Extracellular matrix structural

protein

Mm00483888_m1 COL1A2procollagen, type I,

alpha 2

Extracellular matrix structural

protein

Mm00491889_m1 COL2A1procollagen, type II,

alpha 1

Extracellular matrix structural

protein

Mm00514535_m1 DCN decorinExtracellular matrix structural

protein

Mm01256734_m1 FN1 fibronectin 1 Extracellular matrix linker protein

Mm99999915_g1 GAPDH

glyceraldehyde-3-

phosphate

dehydrogenase

Dehydrogenase, endogenous

control

Mm00439498_m1 MMP2matrix metalloproteinase

2Metalloprotease

Mm00489637_m1 SMAD3MAD homolog 3

(Drosophila)

Transcription factor / TGF-beta

superfamily

Mm00484738_m1 SMAD6MAD homolog 6

(Drosophila)

Transcription factor / TGF-beta

superfamily

Mm00443375_m1 CASR calcium sensing receptor G-protein coupled receptor

Mm01741771_g1 BGLAP

bone gamma-

carboxyglutamate

protein (osteocalcin)

Calcium ion binding protein

Mm00492555_m1 BSP bone sialoproteinExtracellular matrix structural

protein

Table

Page 19

Figure 1.

A B

C D

FE

100 m 25 m

100 m

100 m 100 m

40 m

Figure

Page 20

Figure 2.

Changes of cell number in 24h

0

0,5

1

1,5

2

2,5

CP25 GYPS PMMA

Fold

incr

ease

of c

ell n

umbe

r **

Changes of cell number in 24h

0

0,5

1

1,5

2

2,5

CP25 GYPS PMMA

Fold

incr

ease

of c

ell n

umbe

r **

Page 21

Figure 3.

A.

ALP activity in cultures of 15 days

0

5

10

15

20

25

30

35

40

CP CP25 GYPS PMMA

***

*

ALP

act

ivity

incu

lture

med

ium

(U/g

pro

tein

)

ALP activity in cultures of 15 days

0

5

10

15

20

25

30

35

40

CP CP25 GYPS PMMA

***

*

ALP activity in cultures of 15 days

0

5

10

15

20

25

30

35

40

CP CP25 GYPS PMMA

***

*

ALP

act

ivity

incu

lture

med

ium

(U/g

pro

tein

)

B.

0

0,001

0,002

0,003

0,004

0,005

0,006

0,007

0,008

CP CP25 GYPS PMMA

* ***

SMAD3 expression in cultures of 15 days

Rel

ativ

ee

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ssio

no

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ne

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oun

tof

GA

PD

H

0

0,001

0,002

0,003

0,004

0,005

0,006

0,007

0,008

CP CP25 GYPS PMMA

* ***

SMAD3 expression in cultures of 15 days

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ativ

ee

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no

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ne

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oun

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GA

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H

Page 22

Figure 4.

0

0,00003

0,00006

0,00009

0,00012

CP CP25 GYPS PMMA† †

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BGLAP

0

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0,002

0,003

0,004

CP CP25 GYPS PMMA

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BSP

0

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0,000003

0,0000045

0,000006

CP CP25 GYPS PMMA† †

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CASR

0

1

2

3

4

CP CP25 GYPS PMMA

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**

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gen

eCOL1A1

**

0

0,06

0,12

0,18

0,24

CP CP25 GYPS PMMA

***

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COL2A1

**

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CP CP25 GYPS PMMA

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pres

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0

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0,2

0,3

0,4

CP CP25 GYPS PMMA

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*

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DCN**

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*

0

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0,005

0,0075

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CP CP25 GYPS PMMA

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SMAD6**

0

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0,00006

0,00009

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CP CP25 GYPS PMMA† †

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ee

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BGLAP

0

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0,00006

0,00009

0,00012

CP CP25 GYPS PMMA† †

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BGLAP

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0,002

0,003

0,004

CP CP25 GYPS PMMA

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BSP

0

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0,002

0,003

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CP CP25 GYPS PMMA

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BSP

0

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0,0000045

0,000006

CP CP25 GYPS PMMA† †

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pre

ssio

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CASR

0

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0,000006

CP CP25 GYPS PMMA† †

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e

CASR

0

1

2

3

4

CP CP25 GYPS PMMA

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eCOL1A1

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1

2

3

4

CP CP25 GYPS PMMA

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eCOL1A1

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CP CP25 GYPS PMMA

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COL2A1

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CP CP25 GYPS PMMA

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COL2A1

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CP CP25 GYPS PMMA

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CP CP25 GYPS PMMA

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CP CP25 GYPS PMMA

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0,3

0,4

CP CP25 GYPS PMMA

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DCN**

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CP CP25 GYPS PMMA

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DCN**

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*

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*

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CP CP25 GYPS PMMA

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SMAD6**

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*

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SMAD6**