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Master's Theses University of Connecticut Graduate School

7-6-2018

Comparison of the Effect of Mineral TrioxideAggregate and Bioceramics Root Repair Materialon Odontoblast Differentiation In VitroSaleh Ashkananiashkananis@vcu.edu

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Recommended CitationAshkanani, Saleh, "Comparison of the Effect of Mineral Trioxide Aggregate and Bioceramics Root Repair Material on OdontoblastDifferentiation In Vitro" (2018). Master's Theses. 1256.https://opencommons.uconn.edu/gs_theses/1256

Comparison of the Effect of Mineral Trioxide Aggregate and Bioceramics Root Repair Material on Odontoblast

Differentiation In Vitro

Saleh A. Ashkanani

D.D.S., Virginia Commonwealth University, 2012

A Thesis

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Master of Dental Science

at the

University of Connecticut

2018

Copyright by

Saleh A. Ashkanani

2018

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APPROVAL PAGE

Master of Dental Science Thesis

Comparison of the Effect of Mineral Trioxide Aggregate and Bioceramics Root Repair

Material on Odontoblast Differentiation In Vitro

Presented by

Saleh A. Ashkanani, DDS

Major Advisor _________________________________________________________

Dr. Mina Mina

Associate Advisor_______________________________________________________

Dr. Kamran Safavi

Associate Advisor_______________________________________________________

Dr. Qiang Zhu

University of Connecticut 2018

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Acknowledgements

This project was possible thanks to tremendous effort put on by my major

advisor, Dr. Mina Mina, and the endless support and patience by her lab members, Mrs.

Barbara Rodger and Drs. Anushree Banerjee and Ivana Vidović. The friendly

environment they created in the lab allowed me to learn from my mistakes and

encouraged me to set high expectations and strive to meet them.

I would like to thank my committee advisors, Dr. Kamran Safavi and Dr. Qiang

Zhu, for their support through out the the past three years of my residency. their wisdom

goes beyond dentistry and endodontics. I’m grateful for their insights and

encouragements.

I would like to thank my endodontics family, my co-residents, Dr. I-Ping Chen, Dr.

Blythe Kaufman, Dr. Aniuska Tobin, and Mrs. Erica Zaffetti for their continuous support.

Finally, I must express my very profound gratitude to my wife, Nada, and my

parents, Sara and Adnan for providing me with unfailing support and continuous

encouragement throughout my years of study and through the process of researching

and writing this thesis. This accomplishment would not have been possible without

them. Thank you.

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Table of Contents

Acknowledgment iv

Abstract vi

Chapter I: Introduction— Primary Dentinogenesis ————————————————————————————— — Tertiary Dentinogesis ——————————————————————————————— Chemical Composition of Dentin—————————————————————————— Goal of Vital Pulp Therapy————————————————————————————— Materials for Vital Pulp Therapy —————————————————————————

1 6 8

10 11

Chapter II: Hypothesis and Aims 18

Chapter III: Materials and Methods — Primary Dental Pulp Cultures ——————————————————————————— Preparation of Mineral Trioxide Aggregate and Bioceramics Root Repair Material————— Mineral Detection in Live Cell Cultures———————————————————————— RNA Extraction and Quantitative PCR Analysis———————————————————— Statistical Analysis ———————————————————————————————

2020212223

Chapter IV: Results 24

Chapter V: Discussion 37

Refremces 47

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Abstract

Calcium Hydroxide (CaOH) and Mineral Trioxide Aggregate (MTA) has been used as a

pulp capping material to promote dentinal bridge formation and pulp vitality and healing.

Recently a new material, Bioceramic Root Repair Material (RRM) has been introduced

to the endodontic field. Several studies have indicated similarities in the clinical outcome

of MTA and RRM. However, the underlying mechanisms of action of RRM on dental

pulp cells are still not well studied. The goal of the present study was to examine and

compare the effects of RRM and MTA and on mineralization and expression of selected

markers of odontoblast and osteoblast differentiation using primary dental pulp cells

isolated from un-erupted murine molars. In these studies primary, dental pulp cultures

were indirectly exposed to these materials. The mineralization and expression of

markers were assayed at different stages of differentiation and mineralization using

qPCR and Xylenol orange (XO) staining respectively. Examining of XO staining showed

increase in mineralization in cultured treated with MTA as compared to control and RRM

at day 17 and 21 (P<.05). qPCR analysis showed that MTA increased the expression of

Bsp, Dspp, and Dmp1. On the other hand the levels of Bsp, Dspp, and Dmp1 were

lower in cultures treated with RRM compared to control and MTA-treated cultured. In

conclusion, within the limitation of our study, MTA showed a superior effect on

differentiation of both osteoblast and odontoblasts in primary pulp cultures.

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Chapter I: Introduction

Primary Dentinogenesis

Dentinogenesis is regulated by odontoblasts, highly specialized cells originating from

the cranial neuralcrest-derived cells of the dental papilla. Cranial neural crest from the

areas of midbrain and hindbrain migrate into first branchial arch and give rise to most

connective tissues in the craniofacial region including all components of the teeth,

except the enamel, which is formed by epithelial ameloblasts (Sloan and Smith 2007).

The differentiation of odontoblasts from the neural crest cells is a long process involving

several intermediate steps that are dependent and regulated by sequential signals from

oral epithelium signals (Arana-Chavez and Massa 2004; Lisi et al. 2003; Sloan and

Smith 2007; Thesleff et al. 2001). The first epithelial signals from the early oral ectoderm

lead to the induction of odontogenic potential in the cranial neural crest cells. The next

step in the advancing differentiation within the odontoblast cell lineage is the formation

of dental papilla during the transition from bud to cap stage of tooth development that is

regulated by signals from epithelial bud and primary enamel knot. The primary enamel

knot is a signaling center which forms at the tip of the epithelial tooth bud. It becomes

fully developed and morphologically distinct in the cap stage of tooth development and

expresses at least ten different signaling molecules belonging to the Bone

morphogenetic proteins (BMP), Fibroblast growth factors (FGF), Hedgehog (Hh), and

Wnt signaling pathway (Wnt) families.

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The formation of the dental papilla involves condensation of the odontogenic

mesenchymal cells around the budding epithelium (Nanci 2012). At this stage, the

mesenchymal cells nearest the tip of the epithelial bud give rise to the dental papilla.

The dental papilla cells are the progenitors of odontoblasts, whereas the more

peripheral mesenchyme forms the dental follicle, giving rise to periodontal tissues

(Arana-Chavez and Massa 2004). Next, dental papilla grows rapidly and dental papilla

cells in close proximity to the epithelial-mesenchymal interface at the tip of the cusp

differentiate into pre-odontoblasts, whereas the rest of dental papilla cells from the

dental pulp (Arana-Chavez and Massa 2004; Lisi et al. 2003; Sloan and Smith 2007;

Thesleff et al. 2001).

The differentiation of pre-odontoblasts occurs at the bell stage of tooth development and

is regulated by signals from inner enamel epithelium and secondary enamel knots

(Arana-Chavez and Massa 2004; Lisi et al. 2003; Sloan and Smith 2007; Thesleff et al.

2001). In molar teeth, secondary enamel knots appear in the enamel epithelium at the

sites of the future cusps and express signaling molecules similar to primary enamel

knot. Further differentiation proceeds in a graded fashion from cusp tips towards the

inter-cuspal areas and cervical loops and include the withdrawal of pre-odontoblasts

from cell cycle and the formation of polarized odontoblasts from pre-odontoblasts in

close contact with the epithelial–mesenchymal interface (Sloan and Waddington 2009).

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It has been suggested that pre-odontoblasts located away from the interface becomes

incorporated within the Höhl layer with essential roles in reparative dentinogenesis

(Goldberg and Smith 2004). The formation of pre-odontoblasts is followed by the

formation of polarizing odontoblasts, secretory/functional odontoblasts, and finally the

formation of mature and terminally differentiated odontoblasts (Arana-Chavez and

Massa 2004; Lisi et al. 2003; Sloan and Smith 2007; Thesleff et al. 2001).

Secretory/functional odontoblasts (also called young odontoblasts) are engaged in the

secretion of unmineralized pre-dentin matrix, composed primarily of type I collagen

(Col1a1) (Lisi et al. 2003; Qin et al. 2007). As these odontoblasts continue their

differentiation, they secrete many non-collagenous proteins (NCPs) essential for the

mineralization of collagen fibers and crystal growth (Qin et al. 2004). The NCPs of

dentin include proteins that are also found in bone such as decorin (Steinfort et al.

1994), biglycan (Steinfort et al. 1994), osteonectin (Bronckers et al. 1987; Reichert et al.

1992), and osteocalcin (OC) (Bronckers et al. 1987). Other NCPs in dentin belong to the

SIBLING (small integrin-binding ligand, N-linked glycoprotein) family that includes

osteopontin (OPN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP),

and matrix extracellular phosphoglycoprotein (MEPE) (Qin et al. 2004). High levels of

expression of Dspp and Dsp are the hallmark of odontoblast differentiation and are

routinely used to distinguish differentiated odontoblasts from undifferentiated

progenitors and osteoblasts (Liu et al. 2006). Dspp expression is first detected in

secretory odontoblasts and increases in terminally differentiated odontoblasts. After

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production of mineralized dentin, odontoblasts recede towards pulp and leave behind

cell processes that extend into the mineralized dentin and give dentin matrix its

characteristic tubular morphology (Arana-Chavez and Massa 2004; Lisi et al. 2003;

Sloan and Smith 2007). In mice, the steps between the formation of pre-odontoblasts

and mature odontoblasts are completed within 6–10 hours (Lesot et al. 2001; Ruch et

al. 1995).

Dentin secreted by odontoblasts until the completion of root formation is defined as

primary dentin and forms at a rate of 4–8 μm/day, Following primary dentinogenesis,

odontoblasts remain functional and secrete secondary dentin laid down after the

complete eruption of the tooth into occlusion (Lisi et al. 2003; Sloan and Smith 2007).

Secondary dentin is secreted throughout life at a much slower rate than primary dentin

0.5 μm/day and results in a decrease in the size of the pulp chamber. Histologically and

physiologically, formation of secondary dentin is achieved essentially the same way as

that of primary dentin, though at a much slower rate (~10 times), and it is higher in the

areas subjected to more intense stimuli.

Primary and secondary dentin secreted by odontoblasts, are characterized by closely

packed dentinal tubules that span the entire thickness of the dentin (Sloan and Smith

2007; Thesleff et al. 2001). The major difference between primary and secondary dentin

is the S-curve of the dentinal tubules, which is more pronounced in the secondary

dentin, where odontoblasts located at the periphery of a withdrawing pulp become

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gradually restricted in space (Goldberg et al. 2011). Another difference is the

morphology of odontoblasts themselves engaged in the production of primary and

secondary dentin (Couve and Schmachtenberg 2013).

Thus after their full differentiation, odontoblasts are long-living post-mitotic cells that

align along the dentin-pulp interface, where they maintain pre-dentin and dentin

apposition throughout the whole life of a tooth (Nanci 2012). Odontoblasts are similar to

neurons and cardiomyocytes in that they are basically stable and are not replaced. They

extend cytoplasmic processes into the dentinal tubules in dentin and form a single layer

of tall columnar and highly polarized cell bodies. The number of dentinal tubules is

estimated to be in the range of 60.000-75.000/mm2 in coronal dentin and

20.000-30.000/mm2 in root dentin, as they correspond to the number of odontoblasts. In

addition, odontoblasts in the crown are larger and columnar, whereas they become

smaller and flatter towards the root (Goldberg et al. 2011).

Each odontoblast has a single eccentrically located large nucleus in its basal region.

Mature odontoblasts contain rough and smooth endoplasmic reticulum, Golgi

apparatus, and mitochondria and synthesize various proteins related to dentinogenesis.

Gap junctions between odontoblasts, which mainly consist of connexin 43, might be

involved in the transduction of signals from stimulated odontoblasts to the surrounding

odontoblasts.

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Tertiary Dentinogenesis

The dentin-pulp complex has a regenerative potential leading to the formation of tertiary

dentin. In addition, dentin acts as a physiological barrier that protects the dental pulp

from exogenous noxious stimuli, and the thickness of the dentin barrier can

be increased via the formation of tertiary dentin that forms in response to external

stimuli.

Mild stimuli (irritation, attrition, restorative procedure, deep caries, etc.), lead to

reactionary dentinogenesis during which preexisting odontoblasts up-regulate their

secretory activity (odontoblast-mediated tertiary dentinogenesis). Reactionary dentin

appears either as a layer of osteo-dentin, or as a tubular or atubular orthodentin,

depending on the speed and severity of the stimuli, the progression of the reaction and

the age of the patient (Goldberg et al. 2011). Typically, the presence of reactionary

dentin represents the up regulation of the secretory activity of odontoblasts during mild

adverse stimuli to the tooth (Smith et al. 2003).

However, trauma of greater intensity that causes the death of the pre-existing

odontoblasts leads to reparative dentinogenesis involving the recruitment and

proliferation of progenitor cells to the site of injury and their differentiation into a second-

generation of odontoblasts or “odontoblast-like cells. These odontoblast-like cells

�6

synthesize a dentin-like mineralized tissue, the reparative dentin also referred to as

osteo-dentin, immediately below the site of damage to preserve pulp vitality. These

odontoblast-like are newly formed cells that have similar features to that of odontoblast

cells, and differentiate from the odontoprogenitor cells (progenitor-mediated tertiary

dentinogenesis). However, the exact origin of these progenitor cells are still elusive.

The production of tertiary dentine occurs at a much slower rate than that of primary or

secondary dentin. Reparative dentine is irregular and amorphous, and contains fewer

dentinal tubules than primary or secondary dentin (Nanci 2012). It withholds a particular

structural organization, and has a distinctive chemical composition (Nanci 2012). During

its formation, production of Type I collagen, dentin sialoprotein (DSP) and dentin matrix

protein 1 (DMP1) appears to be down-regulated. On the other hand, the expression of

bone sialoprotein (BSP) and osteopontin are up-regulated (Nanci 2012), resembling the

chemical composition of bone.

It is noted that in clinical conditions, both reactionary and reparative dentin are

compromised by the tertiary dentin formed at the dentin-pulp (Tziafas et al. 1995).

Therefore, reparative dentinogenesis exhibits a much more complex sequence of

biological events, involving recruitment and differentiation of odontoprogenitor cells as

well as up-regulation of secretory activity of odontoblasts, as compared to reactionary

dentin (Smith 2002). It is still unclear which cell type contributes to the formation of

newly formed odontoblast-like cells (Koussoulakou et al. 2009) , and which cellular and

�7

molecular structures are involved in this arrangement, even though reparative

dentinogenesis requires the recruitment of progenitor cells and their differentiation into

odontoblast-like cells during pulp healing.

As primary odontoblasts are postmitotic cells, they are unable to regenerate by cell

division upon damage, implying that there are cells responsible for repair other than

mature odontoblasts. Studies have shown that dental pulp cells at the apex of the

room can serve as a critical source of cells involved in reparative dentinogenesis (About

and Mitsiadis 2001). Furthermore, cell types such as undifferentiated mesenchymal

cells in the pulp core or perivascular progenitors/pericytes, could be involved in this

process (Tziafas et al. 1995). The resulting cell phenotype and its relationship to the

odontoblast can possibly be affected as the embryonic derivation of there progenitor

stem cell is different (Mitsiadis and Rahiotis 2004)

Chemical Composition of Dentin

Mature dentin is represented by approximately 70% inorganic (mineral) material, 20%

organic material and 10% water by weight (40-45%, 30% and 20-25% by volume,

respectively). The composition of dentin makes it harder than bone and softer than

enamel (Goldberg et al. 2011).

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Inorganic component of dentin is primarily composed of hydroxyapatite (HA)

[Ca5(PO4)3OH] (Goldberg et al. 2011). The mineral phase appears first within the

matrix vesicles as single crystals seeded by phospholipids of the vesicle membrane.

These crystals grow rapidly and later fuse with adjacent crystals to form a continuous

layer of the mineralized matrix. Following mineral seeding, NCPs produced by

odontoblasts regulate mineral deposition (Goldberg et al. 2011).

Organic component consists mainly of Type I collagen, which constitutes approximately

90% of all dentin proteins, and is one of the earliest markers of odontoblast

differentiation (Goldberg et al. 2011).

Type I collagen by itself does not induce mineralization, however it serves as an organic

scaffold to further retain NCPs and accommodate a large proportion (~56%) of the

mineral in the holes and pores of the fibrils (Huq et al. 2005). In addition to Type I

collagen, small amounts of Types III and V collagen (~3%) are also present in dentin of

very young animals or during defective collagen synthesis (Goldberg et al. 2011).

Besides Type I collagen, dentin contains various NCPs, including proteoglycans,

glycoproteins, serum proteins, enzymes and growth factors. However, a family of so-

called SIBLING (Small Integrin-Binding Ligand N-linked Glycoprotein) proteins

represents the most abundant group of NCPs. The SIBLING proteins themselves are a

part of a larger family called secretory calcium-binding phosphoproteins (SCPPs), which

�9

are involved directly in binding of calcium ions. There are five members in the SIBLING

family: dentin sialophosphoprotein (Dspp, encoding DSPP protein), dentin matrix protein

1 (Dmp1, encoding DMP1 protein), integrin-binding sialoprotein (IBSP) encoding BSP

protein), secreted phosphoprotein 1 (Spp1, encoding Osteopontin protein) and matrix

extracellular glycophosphoprotein (Mepe, encoding MEPE protein) (Kawasaki and

Weiss 2008)

Goal of Vital Pulp Therapy

Dentists have been using the pulp capping procedure as the treatment of choice to

maintain dental pulp vitality in cases where the pulp is exposed due to caries or trauma.

The treatment consists of sealing the exposed pulp with dental material to facilitate

formation of reparative dentin. The goal of this pulp capping material is to help induce

the differentiation of pulp cells (Takita et al. 2006). The process of pulp tissue healing

includes odontoblast activation and dentinal bridge formation at the exposed site of

dental pulp tissue, and the undifferentiated cells may replace necrotic and dead

odontoblasts (Schroder 1985).

When the dentin-pulp complex is under a greater intensity it causes the death of the

pre-existing odontoblasts which leads to reparative dentinogenesis involving recruitment

of progenitors cells to the site of injury, proliferation, and differentiation into a second

generation of odontoblasts or “odontoblast-like cells” (Sloan and Smith 2007; Sloan and

�10

Waddington 2009). These odontoblast-like cells preserve the pulp vitality by

synthesizing reparative dentin immediately below the site of the damage (Sloan and

Smith 2007; Sloan and Waddington 2009). Available evidence suggests that reparative

dentinogenesis involves several niches of potential progenitor/ stem cells contained in

the pulp (Sloan and Smith 2007; Sloan and Waddington 2009).

Materials For Vital Pulp Therapy

Mineral Trioxide Aggregate (MTA)

Mineral Trioxide Aggregate , first introduced by Torabinejad in the early 1990s, Mineral

Trioxide Aggregate is classified in type-1 Portland cement (PS) category combined with

4:1 ratio of bismuth oxide to provide radiopacity according to the American Standards

for Testing Materials (ASTM). The main component of Portland cement material consists

of tricalcium silicate, dicalcium silicate, tricalcium aluminate, tricalcium oxide, silicate

oxide, aluminoferrite, and gypsum.

Mineral Trioxide Aggregate has excellent sealing ability when used as a sealing material

on accidental perforations or as a root-end filling material (Kennethweldonjr et al. 2002).

Many in vivo and ex vivo studies have shown that Mineral Trioxide Aggregate has

excellent biocompatibility (Torabinejad 1995). Additionally, when Mineral Trioxide

Aggregate was used for direct pulp capping, it showed better interaction with dental pulp

tissue than did calcium hydroxide or acid-etched dentine bonding (Dominguez et al.

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2003). Newly formed hard tissues were observed in direct contact with Mineral Trioxide

Aggregate in cases of pulp capping in-vivo (Andelin et al. 2003).

Comparing white Mineral Trioxide Aggregate (WMTA) with beta-tricalcium phosphate

cement, Dycal, and white Portland cement (WPC) as a pulp capping agent in primary

pirg teeth. Histological examination showed no significant difference in inflammatory cell

response, tissue disorganization, hard tissue formation and bacteria presence between

any of the materials (Shayegan et al. 2009). In another study comparing Mineral

Trioxide Aggregate and calcium hydroxide as pulp capping agents on monkeys’ teeth.

All if the teeth capped with Mineral Trioxide Aggregate showed calcified bridge formation

after 5 months, and the majority were free of inflammation. In contrast, the pulp of teeth

that were capped with calcium hydroxide showed presence of inflammation and

significantly less calcified bridge formation (Ford et al. 1996).

In a study comparing bone morphogenetic protein-7 (BMP-7) and Mineral Trioxide

Aggregate as pulp capping agents on rats’ maxillary molar. The specimens were

immunohistochemically stained for identification of dentin sialoprotein (DSP) staining as

a marker for functional odontoblasts. Histological examination showed that all Mineral

Trioxide Aggregate-capped teeth formed hard tissue that resembled tertiary dentin and

were significantly more positive for DSP in comparison with BMP-7 specimens after two

weeks. The majority of the teeth that were capped with BMP-7 showed formation of

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bone-like hard tissue at the site of pulp capping, but Mineral Trioxide Aggregate-capped

pulps showed significantly more complete dentin bridge formation (Andelin et al. 2003).

In a case report Mineral Trioxide Aggregate was used for pulp capping on a first

primary mandibular molar. There were no pathological findings either on a radiograph

taken after clinical examination after 18 months, and first mandibular primary molar

remained vital after capping with Mineral Trioxide Aggregate (Bodem et al. 2004).

In a case series Mineral Trioxide Aggregate was used for pulp capping or pulpotomy

procedures on 21 teeth. They were followed up monthly for 5 months before they were

extracted and examined histologically. Most pulps responded favorably from a clinical

perspective although a variety of responses were noted histologically including normal

odontoblasts, irregular odontoblasts, intra-pulpal calcifications, dentinal bridges,

cementum formation, internal resorption, inflammatory infiltrate and pulp necrosis.

Fourteen intact third maxillary molar teeth that required extraction were capped with

either Mineral Trioxide Aggregate or calcium hydroxide after inducing standard pulp

exposures. Histologic examination of these teeth at different time intervals showed

dentinal bridge formation and mild chronic inflammation two months after pulp capping

with mineral trioxide aggregate. Specimens treated with calcium hydroxide exhibited the

presence of irregular and thin dentin bridge formation after 3 months, with associated

pulpal necrosis, hyperemia, and inflammation. The authors concluded that Mineral

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Trioxide Aggregate is a better material than calcium hydroxide for treatment of

mechanical pulp exposures (Aeinehchi et al. 2003).

In a case series study, Teeth diagnosed with reversible pulpits were capped using

Mineral Trioxide Aggregate and were followed up for 24 months. Ninety three percent of

the teeth showed clinical and radiographic success after 24 months (Farsi et al. 2007).

In summery, Mineral Trioxide Aggregate has good biocompatibility (Zhu et al. 2014), no

cytotoxicity (Guven et al. 2011), good cell viability (Machado et al. 2016) , good

cytocmpatibility (Zhu et al. 2014), and good sealability (Parirokh and Torabinejad 2010).

However, MTA had drawbacks that includes a long setting time, It causes discoloration

and it is hard to handle clinically (Kohli et al. 2015).

Bioceramics Root Repair Material (RRM):

Recently, a bioceramic material, EndoSequence Bioceramics Root Repair Material

(RRM; Brasseler USA, Savannah, GA), was introduced to endodontic practice. The

main components of bioceramic root repair material are calcium silicates, zirconium

oxide, tantalum pentoxide, and monobasic calcium phosphate. Calcium silicate cements

were shown to promote cell differentiation ( Shie et al. 2012), to have osteoconductive

effects (Chen et al. 2009), and also to reduce inflammation of human dental pulp cells

(hDPCs) (Hung et al. 2014). The suggested clinical applications of Bioceramics Root

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Repair Material are similar to those of Mineral Trioxide Aggregate. Bioceramics root

repair material is seen as a clinically valuable alternative to Mineral Trioxide Aggregate

because of the differences in nanostructure and easier manipulation by the dental

clinician (Leal et al. 2013). Moreover, compared with gray or white Mineral Trioxide

Aggregate, Bioceramics Root Repair Material showed a reduced risk of tooth

discoloration (Kohli et al. 2015). The chemical composition of bioactive materials can

influence the cell behavior, such as differentiation, proliferation, adhesion, or

morphology (Shie and Ding 2013). However, despite significant literature supporting

Mineral Trioxide Aggregate excellent biocompatibility and minimal cytotoxicity, there are

still lack of adequate information Bioceramics Root Repair Material (Chen et al. 2016)

In a study comparing Bioceramics Root Repair Material and Mineral Trioxide Aggregate

effects on dental pulp cells (DPCs) in vitro. The results showed that DPCs had similar

percentages of viable cells when grown on either material. Furthermore, the proliferation

rates, secretion of VEGF, and levels of ALP were similar for DPCs when cultured on

either Bioceramics Root Repair Material or Mineral Trioxide Aggregate (Machado et al.

2016).

Comparing Bioceramics Root Repair Material and Mineral Trioxide Aggregate effects on

pH changes in simulated root resorption defects over 4 weeks in matched pairs of

human teeth. The pH in root surface cavities was measured at various time intervals 20

minutes to 4 weeks. There were no differences in pH between Mineral Trioxide

�15

Aggregate and Bioceramics Root Repair Material at 24 hours at 5mm. The pH of

Mineral Trioxide Aggregate was higher than Bioceramics Root Repair Material by 24

hours at the 2-mm level and after 1 week at the 5-mm level and was thereafter always

significantly higher in white Mineral Trioxide Aggregate compared with Bioceramics Root

Repair Material (Hansen et al. 2011).

Comparing the cytotoxicity and cytokine expression profiles of Bioceramics Root Repair

Material and Mineral Trioxide Aggregate using osteoblast cells (MG-63). Results

showed that Bioceramics Root Repair Material and Mineral Trioxide Aggregate exhibited

minimal levels of cytotoxicity; however, Bioceramics Root Repair Material was slightly

more cytotoxic although not statistically significant. The expression of IL-1β, IL-6, and

IL-8 was detected in all samples with minimal TNF-α expression (Ciasca et al. 2012).

In a study evaluating cytotoxicity and alkaline phosphatase (ALP) activity of Bioceramics

Root Repair Material, and comparing these characteristics with those of Mineral Trioxide

Aggregate and Geristore. The results revealed that the bioactivity of the cells as well as

ALP activity were significantly decreased after exposure to Bioceramics Root Repair

Material elutes in almost all time periods, both in 100% and 50% concentrations, with

the exception of ALP activity of day 1 elutes of bioceramics root repair material at 50%

concentration. Mineral Trioxide Aggregate did not change the bioactivity or ALP activity

of the cells. Geristore elutes of 100% concentration reduced the bioactivity on days 1

and 3, whereas Geristore elutes of 50% concentration affected the cells only on day 1.

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None of the Geristore elutes had any effect on ALP activity of the cells (Modareszadeh

et al. 2012).

In summery, Bioceramics Root Repair Material had similar biocompatibility, cell viability,

PH, and cytotoxicity to MTA. Bioceramics Root Repair Material was easier to handle

clinically and showed minimal discoloration when compared to MTA. However, there is

limited literature about the effect Bioceramics Root Repair Material has on cells

proliferation and differentiation and the available literature is inadequate and

controversial.

�17

Chapter II: Hypothesis:

The similarities in the clinical outcomes of Bioceramics Root Repair Material and

Mineral Trioxide Aggregate on dental pulp and dental bridge formation in vivo is due to

the similarities on their effects on mineralization and differentiation by dental pulp

progenitors. To test this hypothesis, the following two specific aims have been

proposed.

Specific Aims:

Aim 1: Examine and compare the effect of Bioceramics Root Repair Material and

Mineral Trioxide Aggregate on mineralization of dental pulp in vitro.

Primary pulp cultures were prepared as exposed to 20 mg of ProRoot Mineral Trioxide

Rggregate or 20mg of Bioceramic Root Repair Material through transfilters. The effects

on mineralization were examined in live cultures by . Xylenol orange (XO) staining. The

mean fluorescence intensity of XO staining was compared between controls and treated

cultures.

Aim 2: Comparing the effect of Mineral Trioxide Aggregate and Bioceramics Root

Repair Material on the expression of markers of odontoblast and osteoblasts

differentiation in dental pulp in vitro.

To gain a better understanding of possible differences in the effects of Mineral Trioxide

Aggregate and Bioceramics Root Repair Material on odontoblasts and osteoblasts

�18

differentiation, the expression of markers of odobtoblast differentiation (Dspp, Dmp1)

and osteoblast differentiation (Bsp) were examined and compared at various time

points.

�19

Chapter III: Materials and Methods:

Primary Dental Pulp Cultures:

All experimental protocols involving animal tissues were approved by the Institutional

Animal Care and Use Committee, University of Connecticut Health Center. The coronal

portions of the pulps from the first and second molars were isolated from 5- to 7-day-old

murine pulps as described previously (Balic and Mina 2010). After isolation, 2.5 X105

cell/12 well were seeded and grown first in Dulbecco’s modified Eagle’s medium, 20%

fetal bovine serum (FBS), 2 mM L-glutamine, 40 U/ml penicillin, 40 μg/ml streptomycin

and 0.1 μg/ml Fungizone (Invitrogen). Three days later, the medium was changed to

Dulbecco’s modified Eagle’s medium containing 10% FBS. At day 7, mineralization was

induced by addition of Minimum Essential Medium alpha (αMEM) medium, 10% FBS,

with 50 μg/ml fresh ascorbic acid and 4 mM β-glycerophosphate. The medium was

changed every other day.

Preparation of MTA and Bioceramics Root Repair Material:

Fresh ProRoot Mineral Trioxide Aggregate (Dentsply, Tulsa Dental, Tulsa, OK) and pre-

mixed Bioceramic Root Repair Material (Brasseler USA, Savannah, GA) specimens

were prepared according to manufacturer’s guidelines under sterile conditions. The

specimens were allowed to solidify in a humidified CO2 incubator at 37°C for 24 h

before use. 20 mg of ProRoot Mineral Trioxide Aggregate Or Bioceramics Root Repair

Material was placed over permeable and porous membrane inserts measuring 12mm in

�20

diameter (8.0 um pore size) (Corning, New York, NY) to prevent direct physical

interaction between Mineral Trioxide Aggregate /Bioceramics Root Repair Material

specimens and cells, as described. The Inserts were placed over the cells on Day 3.

Mineral Detection In Live Cell Cultures:

Xylenol orange (XO) was added to living cultures in vitro to mark the mineralized

nodules. XO produces a red color when visualized under the microscope using a TRITC

filter. XO powder was being dissolved in distilled water and filtered to make the

concentrated 20 mM stock solution and stored at 40C. XO was added to cultures at a

final concentration of 20 mM for 4 h to overnight. XO-containing medium was replaced

by fresh medium prior to photography to avoid a fluorescent background. The mean

fluorescence intensity of XO staining was measured using a multi-detection

monochromator microplate reader (Safire2, Tecan, Research Triangle Park, N.C., USA)

as described previously (Kuhn, Liu, et al. 2010) with minor modifications. Fluorometric

measurements were performed at 570/610 nm wavelength (excitation/emission) and a

gain of 50. The entire area of each well was read at a scan density of 6 × 6 regions

(high-sensitivity flash mode). The background fluorescence values were subtracted from

respective XO measurements. Measurements were done at Day 10, 14, 17, 21. Four

independent experiments of duplicates were completed.

�21

RNA Extraction and Quantitative PCR Analysis:

Total RNA was isolated using TRIzol reagent (Invitrogen) treated with RNase-free

DNase to eliminate genomic DNA. Isolated RNA was reverse transcribed by Superscript

II Reverse Transcriptase with Oligo(dT)12–18 primers (Life Technologies, Grand Island,

NY). Gene expression in cultured cells was examined by qPCR. TaqMan qPCR

reactions, 9 ng of cDNA was combined with 5 μl TaqMan Universal PCR Master Mix

(Applied Biosystems, Branchburg, NJ), 2.5 μl H2O and 0.5 μl TaqMan primers (total 10

μl). TaqMan primers for Bsp, Dmp1, Dspp, and Gapdh, were purchased from Applied

Biosystems (Table 1). All qPCR reactions were run using 7900HT fast real-time PCR

system (Applied Biosystems) under the following conditions: 50°C for 2 min, 95°C for 10

min, 40 cycles with denaturation at 95°C for 15 s and extension at 60°C for 1 min.

Amplification efficiencies were determined using internal standard curves derived from a

purified amplicon diluted 2-fold (0.14–9.0 ng), and were close to 100% for all qPCR

reactions. We defined the acceptable range of CT values representing gene expression

to be between 10 and 35 cycles, according to the manufacturer’s recommendations

(Applied Biosystems).

�22

Statistical Analysis:

Statistical analysis of the data was performed by GraphPad Prism 7 software using the

paired two-tailed Student t-test. Values in all experiments represented mean ± SEM of

at least 3 independent experiments, and p ≤ 0.05 was considered statistically

significant.

Tables.

Table 1. Primers used to TaqMan qPCR reactions

Gene ID Assay ID

Bsp Mm01208381_g1

Dmp1 Mm00803831_m1

Dspp Mm00515666_m1

Gapdh Mm99999915_g1

�23

Chapter IV: Results

Effects of Mineral Trioxide Aggregate and Bioceramics Root Repair Material on

Mineralization in Primary Dental Pulp Cultures

Previous studies showed that when placed in primary culture, pulp cells from un-erupted

molars proliferated rapidly and reached confluence around day 7 (proliferation phase of

in vitro growth). Following addition of the mineralization-inducing medium at day 7,

these cells underwent differentiation and gave rise to an extensive amount of

mineralized matrix (differentiation/mineralization phase of in vitro growth). The first sign

of mineralization appeared around day 10 with significant increases in the extent of

mineralization thereafter. At day 21 almost the entire culture dish was covered with a

sheet of mineralized tissue

Using this well-characterized dental pulp culture system, we examined the effects of

Mineral Trioxide Aggregate and Bioceramics Root Repair Material on mineralization and

dentinogenesis of primary pulp cultures in vitro. In these experiments, primary pulp

cultures were exposed to Mineral Trioxide Aggregate and Bioceramics Root Repair

Material by placing these over permeable and porous membrane inserts (8.0 um pore

size) to prevent direct physical interaction between materials and pulp cells. Therefore,

controls in these experiments included examining the effects of placement of inserts

without materials (Empty Insert) on mineralization, osteogenesis and dentinogenesis of

primary pulp cultures.�24

XO staining (Figure 1,Table 2) showed a decrease in the extent of mineralization in

cultures with an empty insert, as compared to control at all time points. XO staining also

also showed XO staining showed marked increases in the extent of mineralization in

primary cultures treated with Mineral Trioxide Aggregate at days 17 and 21 as

compared to empty inserts. In cultures treated with Mineral Trioxide Aggregate, the

extent of mineralization reached values similar to cultures without insert. The extent of

mineralization in cultures treated with Bioceramics Root Repair Material was similar to

cultures with empty insert and lower than cultures treated with Mineral Trioxide

Aggregate and no inserts at all time points. These observations showed the positive

effects of Mineral Trioxide Aggregate on mineralization as compared to Bioceramics

Root Repair Material.

Effects of Mineral Trioxide Aggregate and Bioceramics Root Repair Material on

Expression of Markers of Mineralization and Dentinogenesis in Primary Dental

Pulp Cultures

Quantitative PCR analysis showed continuous increases in the expression of all

markers of mineralization and dentinogenesis in control cultures grown with no inserts

between days 7-21 (Figure 2-5 and Table 3-5).

�25

Next, we examined the effects of these materials on the expression of Dspp, shown to

be expressed by odontoblasts but not osteoblasts. qPCR showed marked increases in

the levels of Dspp in cultures treated with Mineral Trioxide Aggregate as compared to

cultures growth with empty inserts and no inserts at days 17 and 21 (Figure 2 and Table

3-5). These values were statistically significant at day 21 (P<.05). On the other hand, in

cultures treated with Bioceramics Root Repair Material, the levels of Dspp were

significantly lower than the levels in all other groups at all time points. These

observations suggest positive effects of Mineral Trioxide Aggregate on the expression of

Dspp as compared to Bioceramics Root Repair Material.

Cultures treated with empty inserts showed decreases in the levels of Dmp1, a known

marker for functional odontoblasts and osteoblasts/osteocytes (Qin et al. 2007) as

compared to cultures grown with no insert (Figure 3 and Table 3-5). Levels of Dmp1, in

cultures treated with Mineral Trioxide Aggregate were higher than the cultures grown

with empty insert and lower than cultures grown without inserts . The changes in the

levels of Dmp1 in cultures treated with Mineral Trioxide Aggregate were statistically

significant at days 17 and 21 (p<.05).

Cultures treated with Bioceramics Root Repair Material showed marked decreases in

the levels of Dmp1 at all time points as compared to cultures treated with Mineral

Trioxide Aggregate as well as cultures grown without insert and empty inserts (Table 3

and Figure 3-5). The decrease of gene expression was statistically significant at days

�26

14 and 21 (p<.05). These observations suggest the positive effects of Mineral Trioxide

Aggregate but not Bioceramics Root Repair Material on the expression of Dmp1.

Cultures treated with empty inserts showed slight increases in the levels of Bsp, which

shown to be expressed by osteoblasts but not odontoblasts, at days 17 and 21 (Figure

4 and Table 3). There was a slight increase of Bsp expression in the cultures treated

with Mineral Trioxide Aggregate as compared to cultures treated with empty inserts

(Figure 4 and Table 3). The levels of Bsp in cultures treated with Mineral Trioxide

Aggregate were higher than the levels in cultures grown without inserts. Unlike in

cultures treated with Mineral Trioxide Aggregate, cultures treated with Bioceramics Root

Repair Material showed decreases in the levels of Bsp at all time points as compared to

all other groups (Figure 4 and Table 3-5). These observations suggest positive effects of

Mineral Trioxide Aggregate but not Bioceramics Root Repair Material on the expression

of Bsp.

�27

Figures.

Figure 1. Comparison of the effects of Mineral Trioxide Aggregate and Bioceramics Root Repair Material on the extent of mineralization in primary dental pulp cultures. The intensity of fluorescence of XO staining at various time points. Results represent mean ± SEM of at least three independent experiments; *p ≤ 0.05 relative to control at each time point.

�28

XO Orange

Days in Culture

Fold

Cha

nge

D10 D14 D17 D210

50

100

150No Insert

Empty Insert

MTA

RRM*

*

* * **

*

Figure 2. Comparison of the effects of Mineral Trioxide Aggregate and Bioceramics Root Repair Material on the expression of Dspp in primary pulp cultures. Primary dental pulp cultures derived from 5-7-day-old pups were established and grown in the absence of insert, with empty insert, an insert with Mineral Trioxide Aggregate and an insert with Bioceramics Root Repair Material between days 3-21 as described in the Materials and Methods. At various time points cells were harvested and processed for RNA isolation and qPCR analysis. Expression levels were normalized to levels at day 10, which is arbitrarily set to 1and is indicated by the dashed line. Results represent mean ± SEM of at least three independent experiments; *p ≤ 0.05 relative to control at each time point.

�29

*DSPP

D7D10 D14 D17 D21

01

100

200

300

400

500

600

Days in Culture

Fold

Cha

nge

No Insert

Empty Insert

MTA

RRM

*

Figure 3. Comparison of the effects of Mineral Trioxide Aggregate and Bioceramics Root Repair Material on the expression of Dmp1 in primary pulp cultures. Primary dental pulp cultures derived from 5-7-day-old pups were established and grown in the the absence of any insert, empty insert, an insert with Mineral Trioxide Aggregate and an insert with Bioceramics Root Repair Material between days 3-21 as described in the Materials and Methods. At various time points cells were harvested and processed for RNA isolation and qPCR analysis. Expression levels were normalized to levels at day 7, which is arbitrarily set to 1and is indicated by the dashed line. Results represent mean ± SEM of at least three independent experiments; *p ≤ 0.05 relative to control at each time point.

�30

*DMP1

D7D10 D14 D17 D21

01

500

1500

2500

3500

4500

Days in Culture

Fold

Cha

nge

No Insert

Empty Insert

MTA

RRM

*

*

*

*

Figure 4. Comparison of the effects of Mineral Trioxide Aggregate and Bioceramics Root Repair Material on the expression of Bsp in primary pulp cultures Primary dental pulp cultures derived from 5-7-day-old pups were established and grown in the absence of any insert, empty insert, an insert with Mineral Trioxide Aggregate and an insert with Bioceramics Root Repair Material between days 3-21 as described in the Materials and Methods. At various time points cells were harvested and processed for RNA isolation and qPCR analysis. Expression levels were normalized to levels at day 7, which is arbitrarily set to 1and is indicated by the dashed line. Results represent mean ± SEM of at least three independent experiments; *p ≤ 0.05 relative to control at each time point.

�31

*BSP

D7D10 D14 D17 D21

01

100

200

300

400

500

600

Days in Culture

Fold

Cha

nge

No Insert

Empty Insert

MTA

RRM

Tables.

Table 2. Comparison of the effects of Mineral Trioxide Aggregate and Bioceramics Root Repair Material on the extent of mineralization in primary dental pulp cultures.

Results represent mean ± SEM of at least three independent experiments. *p ≤ 0.05 relative to the control at each time point. Fold changes represent the FGF2 value divided by the control value for each time point.

No Insert Empty Insert MTA RRM

D10 9 ± 2 8 ± 4 6 ± 2 10 ± 4

D14 40 ± 9 23 ± 6 32 ± 7 27 ± 10

D17 76 ± 14 40 ± 5 64 ± 19 44 ± 6

D21 107 ± 7 68 ± 11 106 ± 19 74 ± 10

�32

Table 3. Effects of culture condition with no inserts on the expression of markers of mineralization and dentinogenesis primary pulp cultures

ΔCT = CTgene - CTGapdh; -ΔΔCT = -(ΔCTsample – ΔCTcontrol).Positive –ΔΔCT value = increased gene expression relative to VH at day 7. Negative –ΔΔCT value = decreased gene expression relative to VH at day 7. Results represent mean ± SEM of at least three independent experiments; *p ≤ 0.05 relative to the control at each time poin

No Insert AVERAGE CtGAPDHD7 18.82±0.26D10 18.88±0.52D14 18.20±0.61D17 18.22±0.46D21 18.51±1.67

BSP ΔCt ΔΔCt foldchangeD7 24.90±0.37 5.29±0.11D10 21.99±1.25 3.11±0.73 -2.97±0.62 8.18±3.40D14 18.77±2.12 0.57±1.51 -5.51±1.40 56.65±47.65D17 17.95±1.00 -0.27±0.54 -6.35±0.42 83.52±24.23D21 16.20±1.67 -2.31±0.00 -8.40±0.11 337.33±25.85

DMP1 ΔCt ΔΔCt foldchangeD7 31.76±1.47 12.95±1.21 0.00±0.00 1.00±0.00D10 24.11±4.79 5.23±4.28 -7.71±3.06 493.97±632.41D14 21.27±3.40 3.07±2.79 -9.87±1.58 1233.26±1130.88D17 20.31±2.70 2.09±2.24 -10.86±1.02 2095.75±1375.12D21 20.24±4.00 1.73±2.34 -11.21±1.12 2744.10±1942.65

DSPP ΔCt ΔΔCt foldchangeD7D10 36.55±0.45 17.67±0.97 0.00±0.00 1.00±0.00D14 31.36±1.09 13.16±0.48 -4.51±1.45 28.72±24.82D17 29.68±0.42 11.46±0.04 -6.21±0.93 81.73±49.19D21 28.12±0.42 9.62±1.25 -8.05±0.28 268.34±52.26

�33

Table 4. Effects of empty Insert on the expression of markers of mineralization and dentinogenesis in primary dental pulp cultures.

ΔCT = CTgene - CTGapdh; -ΔΔCT = -(ΔCTsample – ΔCTcontrol).Positive –ΔΔCT value = increased gene expression relative to VH at day 7.Negative –ΔΔCT value = decreased gene expression relative to VH at day 7. Results represent mean ± SEM of at least three independent experiments; *p ≤ 0.05 relative to the control at each time point.

EmptyInsert AVERAGECtGAPDHD7 19.41±1.39D10 18.42±1.01D14 18.58±0.49D17 18.39±0.84D21 18.90±1.52

BSP ΔCt ΔΔCt foldchangeD7 25.87±0.51 5.29±1.61D10 23.00±1.28 4.58±0.47 -1.87±1.30 4.80±4.22D14 21.27±2.40 2.69±2.71 -3.76±3.39 35.47±29.97D17 17.94±2.33 -0.45±1.61 -6.90±0.75 130.19±63.46D21 16.89±1.32 -2.01±0.97 -8.46±0.84 396.65±237.18

DMP1 ΔCt ΔΔCt foldchangeD7 31.91±1.07 12.50±1.56 0.00±0.00 1.00±0.00D10 25.71±4.56 7.29±3.67 -5.21±2.95 107.41±149.17D14 22.50±2.99 3.93±3.30 -8.57±3.26 945.03±800.98D17 20.85±2.87 2.47±2.05 -10.03±1.64 1442.02±1015.64D21 20.53±2.94 1.62±1.74 -10.87±0.59 1979.27±756.04

DSPP ΔCt ΔΔCt foldchangeD7 0.00±0.00D10 34.94±1.50 16.52±2.49 0.00±0.00 1.00±0.00D14 33.53±4.09 14.96±4.52 -1.56±6.91 56.85±89.01D17 30.83±2.18 12.44±1.37 -4.08±3.47 88.43±144.75D21 28.82±0.02 9.91±1.51 -6.61±0.99 112.58±67.86

�34

Table 5. Effects of Mineral Trioxide Aggregate on the expression of markers of mineralization and dentinogenesis in primary dental pulp cultures.

ΔCT = CTgene - CTGapdh; -ΔΔCT = -(ΔCTsample – ΔCTcontrol).Positive –ΔΔCT value = increased gene expression relative to VH at day 7.Negative –ΔΔCT value = decreased gene expression relative to VH at day 7. Results represent mean ± SEM of at least three independent experiments; *p ≤ 0.05 relative to the control at each time point.

MTA AVERAGECtGAPDHD7 18.42±1.10D10 19.17±0.98D14 18.13±0.27D17 18.71±1.43D21 18.44±0.90

BSP ΔCt ΔΔCt foldchangeD7 26.65±1.16 5.29±1.29D10 22.93±1.53 3.77±0.99 -4.46±1.60 33.51±36.51D14 21.68±1.61 3.54±1.56 -4.69±2.83 54.29±44.92D17 19.19±0.66 0.49±1.23 -7.74±0.31 217.17±47.35D21 17.97±0.22 -0.47±1.09 -8.70±0.56 437.93±181.69

DMP1 ΔCt ΔΔCt foldchangeD7 31.93±1.10 13.51±1.62 0.00±0.00 1.00±0.00D10 25.92±2.92 6.75±1.94 -6.76±1.95 184.00±203.66D14 23.17±3.13 5.03±3.03 -8.48±3.10 1025.64±1338.65D17 21.03±3.04 2.33±1.87 -11.18±0.66 2473.32±968.82D21 20.43±2.48 1.99±1.69 -11.52±0.34 2980.46±652.47

DSPP ΔCt ΔΔCt foldchangeD7 0.00±0.00D10 38.37±0.58 19.01±1.23 0.00±0.00 1.00±0.00D14 34.41±2.87 16.34±2.72 -2.67±3.94 23.44±27.45D17 30.40±2.23 11.91±2.65 -7.10±2.37 267.23±289.36D21 28.78±0.73 10.29±0.82 -8.72±0.51 439.64±162.14

�35

Table 6. Effects of Bioceramics Root Repair Material on the expression of markers of mineralization and dentinogenesis in primary dental pulp cultures.

ΔCT = CTgene - CTGapdh; -ΔΔCT = -(ΔCTsample – ΔCTcontrol).Positive –ΔΔCT value = increased gene expression relative to VH at day 7.Negative –ΔΔCT value = decreased gene expression relative to VH at day 7. Results represent mean ± SEM of at least three independent experiments; *p ≤ 0.05 relative to the control at each time point.

RRM AVERAGECtGAPDHD7 19.44±0.38D10 18.58±0.94D14 18.41±0.45D17 18.11±0.80D21 18.81±1.52

BSP ΔCt ΔΔCt foldchangeD7 26.11±0.58 5.29±0.88D10 23.15±2.30 4.56±1.36 -2.10±0.59 4.54±1.99D14 20.29±2.16 1.88±2.04 -4.78±1.26 36.04±33.50D17 17.80±1.09 -0.32±0.79 -6.98±0.09 126.13±7.73D21 17.40±1.47 -1.41±0.73 -8.07±0.49 278.93±87.51

DMP1 ΔCt ΔΔCt foldchangeD7 31.38±1.83 11.94±1.95 0.00±0.00 1.00±0.00D10 26.82±5.25 8.24±4.31 -3.70±2.57 27.74±31.50D14 22.74±2.52 4.33±2.22 -7.60±0.51 201.96±63.21D17 20.89±2.94 2.78±2.52 -9.16±0.62 605.88±251.17D21 20.86±4.02 2.05±2.61 -9.88±0.77 1034.49±525.36

DSPP ΔCt ΔΔCt foldchangeD7D10 34.41±1.07 16.02±1.29 0.00±0.00 1.00±0.00D14 31.26±2.53 12.78±2.13 -3.25±3.43 33.09±45.73D17 30.87±1.61 12.54±0.91 -3.48±2.11 21.34±26.48D21 29.83±1.35 11.07±1.30 -4.96±1.28 40.44±35.63

�36

Chapter V: Discussion

Dental Trauma, dental caries, and iatrogenic procedures can lead in pulp exposure.

Recently, dentists are shifting to treat pulp exposures with a vital pulp therapy as they

have became a predictable alternative to pulpectomy (Aguilar and Linsuwanont 2011).

The practice of vital pulp therapy had significant advances in term of techniques and

materials used, and the practices has shifted from the hopeless organ concept to one of

optimistic (Cox et al. 1985). When the exposed pulp is provided with a proper biological

seal and maintained against leakage of oral contaminants, the pulp cells has the

capacity to reorganize and for form a bridge to protect the pulp (Stanley 1989). In a

classic study, pulp exposures were done on conventional rats and germ-free rats. The

germ free animals, the pulp remained vital and and signs for dentinal bridge formation

began at day 14 and was complete by day 28 (Kakehashi et al. 1965). However, the

oral cavity is not a germ-free environment and providing a good seal, among many

other factors, is crucial to help the tooth remain vital and form a dentinal bridge. The

vital pulp therapy is considered successful if the tooth remains vital and a bridge is

formed within 75-90 days (Pameijer and Stanley 1998). The main reasons for a vital

pulp therapy failure and causes of post-operative inflammation and pulp necrosis are

non-sterile procedures and bacterial micro-infiltration of the pulp via dentin tubules

(Stockton 1999).

�37

A tooth pulp has a number of important functions throughout the life a tooth. These

include formation, induction, nutrition, sensation and defense. The pulp retains its ability

to form dentin following tooth development (Fish 1932). Vital pulp tissue contributes to

the production of secondary dentin, peri-tubular dentin and reparative dentin in

response to biologic and pathologic stimuli throughout the life of a tooth (Stanley 1989).

This enables the vital pulp to partially compensate for the loss of enamel or dentin

caused by mechanical trauma or disease (Fish 1932).

A great number of medicaments and materials have been used as vital pulp therapy

agents, i.e. different types of cements, gold and silver, antiseptic pastes, ivory powder,

dentin chips, chemotherapeutics, plaster of Paris, calcium hydroxide, magnesium oxide

and many others (Obersztyn 1966). Some of the most promising materials consisted of

pastes utilizing calcium hydroxide and spicules of dentin. The calcium hydroxide paste

was introduced by Hermann in the 1920s, which marked a new era in dental pulp

therapy by demonstrating that a calcium hydroxide mixture induced bridging of the pulp

surface with reparative dentin (Stanley 1989).

Calcium hydroxide is widely used as a viable material of choice for direct pulp capping.

However, clinical success rates vary considerably from 13% up to 97.8% (Barthel et al.

2000). Examining the clinical data on direct pulp capping with calcium hydroxide or its

compounds showed that success rates decrease as follow-up periods increase. Clinical

trials report that 1–2 years after vital pulp therapy, success rates of more than 90% have

�38

been achieved (Beetke et al. 1990). After 2–5 years, success rates drop from 81.8% to

37% (Auschill et al. 2003). Ten years after vital pulp therapy with a setting calcium

hydroxide paste, treatment outcome is considered successful in only 13% of cases

(Barthel et al. 2000).

Recent attempts to develop more biocompatible pulp capping materials have resulted in

the development of two new materials, Mineral Trioxide Aggregate and bioceramics root

repair material. Mineral Trioxide Aggregate was initially developed to seal off all

pathways of communication between the root canal system and the external surface of

the tooth (Lee et al. 1993). White Mineral Trioxide Aggregate is a powder consisting of

fine hydrophilic particles of tricalcium silicate, dicalcium silicate, tricalcium aluminate,

calcium sulfate dehydrate and bismuth oxide. It also contains small amounts of other

mineral oxides, which modify its chemical and physical properties. The powder consists

of fine hydrophilic particles that set in the presence of moisture. Hydration of the powder

results in a colloidal gel that solidifies to a hard structure. Bismuth oxide powder has

been added to make the aggregate radiopaque. Since its first description in the

literature in 1993, Mineral Trioxide Aggregate has shown numerous exciting clinical

applications in endodontics (Torabinejad and Parirokh 2010). It was used on an

experimental basis for several years with promising results, in both surgical and non-

surgical applications, including root-end fillings, perforation repairs in roots or furcations,

�39

apexification, pulpotomies, and recently as a potential alternative material for pulp

capping (Torabinejad and Parirokh 2010).

Mineral Trioxide Aggregate is increasingly recognized as the preferred choice of

capping/filling material because of its superior biocompatibility and sealing ability.

Animal studies revealed that Mineral Trioxide Aggregate induces calcified barrier

formation in the absence of any noteworthy inflammation (Ford et al. 1996). In addition,

in vitro cell culture studies showed that Mineral Trioxide Aggregate stimulates cell

proliferation (D'Anto et al. 2010). Consistent with these findings, clinical data showed

high success rates associated with the use of Mineral Trioxide Aggregate (Bogen et al.

2008).

In vitro trials and histologic studies report favorable results regarding the chemical and

physical properties, antibacterial activity, biocompatibility, and sealing properties of

Mineral Trioxide Aggregate (Torabinejad and Parirokh 2010). Clinical success rates are

equally promising (Aguilar and Linsuwanont 2011). Comparing treatment outcome of

calcium hydroxide and Mineral Trioxide Aggregate for vital pulp therapy, suggested that

Mineral Trioxide Aggregate might be the material of choice (Cho et al. 2013). The

drawbacks of Mineral Trioxide Aggregate are its high costs, long setting time, and

potential for discoloration of the tooth (Parirokh and Torabinejad 2010).

�40

The shortcomings of Mineral Trioxide Aggregate including difficulties of handling and

long setting time, have led to utilization if a new bioceramic material, Bioceramics Root

Repair Material (RRM; Brasseler USA, Savannah, GA), for viable pulp capping material.

The main components of Bioceramics Root Repair Material are calcium silicates,

zirconium oxide, tantalum pentoxide, and monobasic calcium phosphate. Calcium

silicate cements were shown to have osteoconductive effects (Chen et al. 2009), to

promote cell differentiation (Shie et al. 2012), and also to reduce inflammation of human

dental pulp cells (hDPCs) (Hung et al. 2014).

The suggested clinical applications of Bioceramics Root Repair Material are similar to

those of Mineral Trioxide Aggregate (Chen et al. 2016). Bioceramics Root Repair

Material is seen as a clinically valuable alternative to Mineral Trioxide Aggregate

because of the differences in nanostructure and easier manipulation by the dental

clinician (Leal et al. 2013). Moreover, compared with gray or white Mineral Trioxide

Aggregate, Bioceramics Root Repair Material showed a reduced risk of tooth

discoloration (Kohli et al. 2015). The chemical composition of bioactive materials can

influence the cell behavior, such as differentiation, proliferation, adhesion, or

morphology (AlAnezi et al. 2011). However, although it has been repeatedly shown that

Mineral Trioxide Aggregate has excellent biocompatibility and minimal cytotoxicity, there

are still only a few studies on Bioceramics Root Repair Material with ambivalent

conclusions.

�41

There are only 8 in vitro studies on the cytotoxicity of Bioceramics Root Repair Material,

and the results are variable. Three studies (Ciasca et al. 2012; Hirschman et al. 2012;

Leal et al. 2013) found no difference in cell viability between Bioceramics Root Repair

Material and Mineral Trioxide Aggregate. On the other hand, four studies showed that

Bioceramics Root Repair Material was associated with lower cell viability in some

conditions (Damas et al. 2011; Ma et al. 2011; Modareszadeh et al. 2012; Samyuktha et

al. 2014). One study found no difference in proliferation of periodontal ligament

fibroblasts grown on Bioceramics Root Repair Material versus Mineral Trioxide

Aggregate, whereas osteoblasts showed significantly higher proliferation on

Bioceramics Root Repair Material than Mineral Trioxide Aggregate (Willershausen et al.

2013).

In this study we examined and compared the effect of Bioceramics Root Repair material

and Mineral Trioxide Aggregate on mineralization of dental pulp and expression of

markers of odontoblast and osteoblasts differentiation in primary cultures derived from

the coronal portions of dental pulp from an un-erupted 5-7 days murine molars. The

reagents were introduced into the culture using a transwell insert as described in the

methods section. At the pilot study, we noticed introducing an insert to the cell at day 3,

with or without the reagents, made the cells concentrate at the center of the well instead

of spreading around the well evenly. We attributed that due to the surface tension

created by the transwell insert. Increasing the media to 2 ml instead of the original 1ml

appears to have solved the problem.

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In the first part of the study, we examined the effect Bioceramics Root Repair Material

and Mineral Trioxide Aggregate has on the mineralization of the primary culture. At day

10 we started noticing all group to show some mineralization. we had two control

groups, one without any transwell inserts and the other group had an empty transwell

insert. We noticed at D14, D17 and D21 empty insert had decreased the rate of

mineralization at when compared to No insert (control). Therefore, an empty insert

served as a negative control when comparing Mineral Trioxide Aggregate and

Bioceramics Root Repair Material effect on mineralization. Mineral Trioxide Aggregate

showed statistically significant increase mineralization when compared the control at

day 17 and 21 (p <.05). However, Bioceramics Root Repair Material decreased

mineralization when compared to control at day 21.

The decrease of mineralization in Bioceramics Root Repair Material in our study is

consistent with other studies. In one of the study where they evaluated and compared

the cytotoxicity and effects on ALP activity of Bioceramics Root Repair Material and

Mineral Trioxide Aggregate using a human osteosarcoma cell line. They found that the

effect on bioactivity of the cells as well as ALP activity were significantly decreased after

exposure to Bioceramics Root Repair Material elutes in almost all time periods, both in

100% and 50% concentrations, with the exception of ALP activity of day 1 elutes of

Bioceramics Root Repair Material at 50% concentration. Mineral Trioxide Aggregate did

not change the bioactivity or ALP activity of the cells (Modareszadeh et al. 2012).

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In the second part of the study, we looked at the gene expression of Dmp1, Dspp and

Bsp at day 7, 10, 14, 17, and 21. Mineral Trioxide Aggregate had an positive effect,

increasing the expression of all examined markers compared to control. Bioceramics

Root Repair Material, on the other hand, had a negative effect on gene expression of

Dmp1 and Dspp. This suggests Mineral Trioxide Aggregate induced the formation

osteodentin better than Bioceramics Root Repair Material. Our results are not

consistent with the results reported by others.

A recent study published found Mineral Trioxide Aggregate and Bioceramics Root

Repair Material had an opposing effect on cells proliferation from our results. They had

standardized human dentin disk lumens were filled with Biodentine, Endosequence BC

Root Repair Material-Putty, Endosequence BC Root Repair Material-Putty Fast set, or

Mineral Trioxide Aggregate , and stem cells of the apical papilla (SCAP) were cultured

directly onto the samples. They measures cell viability at 7 days, whereas differentiation

into a mineralizing phenotype was evaluated by real-time reverse-transcription

polymerase chain reaction and alizarin red staining at 21  days in culture. A greater

expression of alkaline phosphatase messenger RNA and Dspp was noted in the

Bioceramics Root Repair Material group, whereas Mineral Trioxide Aggregate

promoted a greater expression of the osteoblastic marker IBSP (Miller et al. 2018).

.

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One Limitation of our study was having to change the media every other day, this might

have caused the dilution of Calcium ions and Silicon ions released from Mineral Trioxide

Aggregate and Bioceramics Root Repair Material to over the period of the experiment.

Another limitation in our study design was although we were able to measure

mineralization using XO staining of the same cells culture over the period of the

experiment, the same couldn’t be done measuring markers expression with qPCR, as

the cells needed to be harvested at different time points, different plates were used for

each time point.

There has been a great advancements in our clinical armamentarium (ie, materials,

instruments, and medications) and knowledge from the trauma and tissue engineering

fields that can be applied to regeneration of a functional pulp-dentin complex.

Despite the progress made in the field of pulp biology, there is no single therapeutic

regimen for direct pulp capping that can achieve, predictably and reliably, the goals of

preserving tooth vitality and tooth function. What is agreed upon is that an effective pulp

capping material should be biocompatible, provide a biological seal, prevent bacterial

leakage and stimulate dentin bridge formation (Koh et al. 1997) Mineral Trioxide

Aggregate showed clinical and radiographic success as a pulp capping agent in

permanent teeth and was found to be as successful as calcium hydroxide when used

for direct pulp capping in teeth (Abedi and Ingle 1995).

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In summary, vital pulp therapy is a very predictable procedure when done in a sterile

environment and utilizing a material that has good sealability and biocompatibility. With

the limitation of our study, we found that Mineral Trioxide Aggregate serves as a

superior material when used for a vital pulp therapy, it increased mineralization and

promoted odontoblast markers when compared to control. Although Bioceramics Root

Repair Material is the preferred material of choice by clinician due to it’s fast set time,

limited tooth discoloration and ease of you, our finding showed it had decreased

mineralization and odontoblast markers expression when compared to control. Further

in vitro and in vivo investigations are required to prove the clinical applications of these

materials.

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