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University of ConnecticutOpenCommons@UConn
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
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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
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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
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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
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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
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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.
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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
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differentiation, the expression of markers of odobtoblast differentiation (Dspp, Dmp1)
and osteoblast differentiation (Bsp) were examined and compared at various time
points.
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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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