THE EFFECT OF MAGNESIUM ON DENTINOGESIS OF HUMAN DENTAL

PULP CELLS

1Department of Restorative Sciences and Biomaterials, Boston University Henry M Goldman School of Dental Medicine2 Department of Endodontics, Boston University Henry M Goldman School of Dental Medicine

Rania Salem1,2, Chang Zhang 1, Dr. Sami Chogle 2, Dr. Laisheng Chou 1

• Introduction• Background• Research Questions• Hypothesis• Research Objectives• Materials and Methods• Results• Conclusions• Clinical Considerations• Future Studies• References

OVERVIEW

• Dental caries is one of the most prevalent chronic diseases of allpopulations worldwide

• According to the report from the National Institute of Dental andCraniofacial Research (NIDCR) 92% of adults aged 20–64 years in theUnited States have had dental caries in their permanent teeth

• Over $ 66 billion is spent annually in the United States because ofuntreated dental caries

(Selwitz et al., 2007)(Data from NIDCR Survey 2018)

• Dental pulp exposure from dental caries can cause pain and infection

• The currently available treatment scenarios in such condition areeither root canal treatment if the pulp is irreversibly inflamed or pulptherapy if the pulp is still vital

• Vital pulp therapy aims to induce dentinal bridge formation to maintainpulp integrity and function

(Renton &Wilson 2016)(Qureshi et al., 2014)

• Maintaining pulp vitality is a major challenge

• The development of pulp capping agents has been instrumental in promotingreparative dentin formation

• However, on‐going research strategies have failed to overcome the limitations ofexisting pulp capping materials so that healthy and progressive regeneration of theinjured tissues is attained

(Qureshi et al., 2014)

• Critical need exists to develop a novel therapy that inducesdentinogenesis and remineralization similar to the naturalprocess

• Introducing therapeutic ions into pulp capping materials hasbeen considered as a new approach for enhancing regenerationof dental tissues.

• Emerging evidence supports a notion that magnesium ions playsindispensable bioactive rolesEmerging evidence supports a notion thatmagnesium ions play indispensable bioactive roles

BACKGROUND

Act as an intracellular second messenger

Connect cell-surface receptor induction & cytosolic effectors

Critical for ATP dependent phosphorylation of DNA,RNA & enzymes

Modulation of signal transduction

Affect energy metabolism

Induce cell proliferation, differentiation & cell matrix interaction

(Hou.etal.,2020)(Alhosani,M&. Leeny 2015)(Wesmann et al., 1997)(Saris et al., 2000)(Serre et al.,1998)(Staiger et al., 2006)

Bone homeostasis

Induce mineral metabolism

Influence bone density & biomechanical property

Associated in biomineralization of bone & teeth

Cofactor for Alkaline phosphatase (ALP)

Induction of osteoblast differentiation &increase in mineralization

Increase in bone formation & cancellous bone volume

(Cashiglioni et al.,2013)(Grober et al.,2015)(Zhang et al.,2017)(Wiesmann et al., 1997Sans et al., 2000)(Burmester et al .,2017 Wu et al .,2015,Kim et al., 2017,Lin et al.,2019, Terranora et al., 2016)(Toba et al., 2000)

Accelerate early osseointegration at titanium/bone interface

Mg phosphate cements promote proliferation & differentiation of bone

marrow stem cells Mg-doped glasses promote biocompatibility & enhance

osteogenic bioactivity Porous magnesium scaffolds

stimulate angiogenesis & induce new bone formation

Mg- screws promote expression of BMP2 & VEGF in bone defects (Galli et al.,

l2014,2015.2017)(Sun et al .,2006Hussain et al .,2002,Wei et al., 2016)(Bob et, al .,2013)(Sun. et,al,., 2006)(Wang et al., 2017)(Cheng .eta.,,2016)

Mg- ceramics enhance differentiation & expression of

osteoblast-related genes

• Evidence in the literature indicates that magnesium ions havea stimulatory effect on normal human osteoblasts

• Magnesium ions represent a promising strategy to replacedamaged bone structures and restore their biological functions

• However, to date the effect of magnesium ions on dentin regeneration is intriguinglargely unexplored

• Because dentin closely resembles bone physically and chemically

The mechanism behind magnesium relationship to human dental pulp cellbehavior is not fully understood !!!!!!

Will magnesium ions have a similar mechanism of action on human dental pulpcells triggering regeneration of the injured/damaged dentin-pulp complex?

• 1- Will magnesium ions have a (stimulatory) biologic effect on human dental pulp cells (HDPCs)in vitro ?

• 2- Whether supplemental magnesium will affect such odontogenic activity enhancing orinhibiting proliferation, differentiation and biomineralization of human dental pulp cells in vitro ?

• 3- What will be the optimal concentration that can mostly induce such dentinogenic effect ?

Research Questions

• Magnesium will have an inductive biologic effect on human dental pulpcells (HDPCs) in vitro

Hypothesis Hypothesis

• 1- To prepare different concentrations of magnesium chloride

• 2- To assess the stimulatory effect of different concentrations of supplemental magnesium onattachment, proliferation, cell viability, and mineralization of human dental pulp cells (HDPCs)in vitro

• 3- To evaluate odontogenic differentiation by measuringo Alkaline phosphatase (ALP) activityo Dentin sialoprotein (DSP)o Dentin matrix protein-1(DMP-1)o Dentin sialo phosphoprotein (DSPP)

Research Objectives

MATERIALS & METHODS

Preparation of Magnesium Chloride

Magnesium Chloride Magnesium

Chloride

G H

Steps of Human Dental Pulp Cells (HDPCS)Extraction

Control MgCl21 mMMgCl2

4 mMMgCl2 MgCl2

0.5 mM MgCl2

2 mM 8 mM

Secondary Passage of Human Dental Pulp Cell Culture

Control Magnesium Chloride(MgCl2)

Outcome measures

Human Dental Pulp Cells Cultured with Magnesium Chloride Concentrations

Attachment Efficiency 4, 8,12,16 and 20 hrs

Proliferation7,10,14 days

Differentiation 7,10,14 days

Mineralization7,10,14 days

ALP DSP DMP1 DSPP

Primary Passage of Human Dental Pulp Cell Culture

Freshly Extracted Third Molar Teeth

RESULTSRESULTS

0

20

40

60

80

100

120

Control 0.5 mm 1mm 2mm 4mm 8mm

Cel

l Atta

chm

ent E

ffeci

ency

(%)

Concentrations of Supplemental Magnesium Chloride (MgCl2) mM

Histogram showing Cell Attachment Efficiency at 16 hours of all Magnesium Chloride (MgCl2) Concentrations

0

5

10

15

20

25

30

35

40

45

50

Day 3 Day 7 Day 10 Day 14 Day 21

Cel

l Pro

lifer

atio

n (F

olds

).

Time (Days)

Control 0.5 mM 1 mM 2 mM 4 mM 8 mM

a aa ab

ba

b b

d d

a

b b

c

c d

a

bb

c

d d

a

b

b

c

d d

c

Histogram showing Cell Proliferation Rate of Magnesium Chloride (MgCl2) at Different Time Intervals

0

20

40

60

80

100

120

16 hrs Day 3 Day 7 Day 10 Day 14

Cel

l Via

bilit

y %

Time (Days)

Control 0.5 mM 1 mM 2 mM 4 mM 8 mM

b cd

ef

a

b c

d

e f

a

b b

c

ef

ab b

ab b c

cd

ab

dd

Histogram showing Cell Viability of Magnesium Chloride (MgCl2) at Different Time Intervals

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Day 7 Day 10 Day 14

Cel

l Pro

lifer

atio

n (F

olds

)

Time (Days)

Control 0.5 mM 1 mM 2 mM 4 mM 8 mM

a

b b ba a

a

bc

a

de

a

b

b

a

a

d

P<0.0001

Histogram showing Cell Proliferation Rate of Magnesium Chloride (MgCl2) at Different Time Intervals(Odontogenic Media)

0

0.5

1

1.5

2

2.5

3

3.5

Day 7 Day 10 Day 14

ALP

Activ

ity (m

U/m

l ) P

er M

illion

Cel

ls

Time (Days)

Control 0.5 mM 1 mM 2 mM 4 mM 8 mM

a b

cd

b

c

d

c

a

a

b b

c

d

e

f

a

b

P<0.0001

Histogram showing Alkaline Phosphatase Activity of Magnesium Chloride (MgCl2) at Different TimeIntervals

0

50

100

150

200

250

300

350

400

450

500

Day 7 Day 10 Day 14Den

tin S

ialo

prot

ein

Expr

essi

on (n

g/m

l) Pe

r Milli

on C

ells

Time (Days)

Control 0.5 mM 1 mM 2 mM 4 mM 8 mM

c da e

a

bc d

e f

a

b ce

f

d

ba

Histogram showing Dentin Sialoprotein (DSP) Expression of Magnesium Chloride (MgCl2) at Different TimeIntervals

0

100000

200000

300000

400000

500000

600000

Day 7 Day 10 Day 14

Den

tin M

atrix

Pro

tein

Exp

ress

ion

(DM

P-1)

(p

g/m

l) Pe

r Mill

ion

Cel

ls

Time (Days)

Control 0.5 mM 1 mM 2 mM 4 mM 8 mM

a

bc d

e

f

a

bd

e

f

a bc d e

f

P<0.0001

Histogram showing Dentin matrix protein1(DMP-1) Expression of Magnesium Chloride (MgCl2)at Different Time Intervals

c

0

50000000

100000000

150000000

200000000

250000000

300000000

350000000

400000000

450000000

500000000

Day 7 Day 10 Day 14

Den

tin S

ialo

pho

spho

(DSP

P) P

rote

in E

xpre

ssio

n (n

g/m

l) Pe

r Milli

on C

ells

Control 0.5 mM 1 mM 2 mM 4 mM 8mM

ec d

e f

f

ab

c

da

b

c

d

b

Histogram showing Dentin Sialophosphoprotein (DSPP) Expression of Magnesium Chloride (MgCl2)at Different Time Intervals

a

e

P<0.0001

Time (Days)

f

0

1

2

3

4

5

6

7

8

9

Day 7 Day 10 Day 14

Min

erliz

atio

n R

ate

per m

illion

cel

ls

Time (Days)

Control 0.5 mM 1 mM 2 mM 4 mM 8 mM

a

b

c

a

b

c

d

e f

d d e

ab

c c c d

Histogram showing Mineralization Rate of Magnesium Chloride (MgCl2) at Different Time Intervals

CONCLUSIONS

•Conclusions

• This is the first report to investigate the optimal magnesiumconcentration needed to induce human dental pulp cellattachment, proliferation, differentiation and mineralization

•Conclusions

• This study showed that HDPCs with 0.5mM and 1 mM magnesiumsupplemental groups elicited the highest stimulatory effect on cell attachmentefficiency, cell viability, proliferation rate, ALP activity, expression ofodontogenic-related proteins (DSP, DMP-1, DSPP) and mineralization at alltime points

Conclusions

• However, 8mM magnesium group had an inhibitory effect on HDPCSbehavior showing lower ALP activity, expression of DSP, DMP-1, DSPP, andmineralization compared to the control

• Future implementation of magnesium containing biomaterialsshow promise as a potential novel dental pulp capping materialor additive in regenerative endodontics

DiscussionClinical Considerations

Future Studies

● Further studies on the role of magnesium and its effect on odontogenic differentiation to determine the associated signaling pathway warrants closer examination

15

Acknowledgements

oDr. Laisheng Chou, AdvisoroDr. Sami ChogleoChang Zhang, Lab ManageroCo-residents

17

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References

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