Molecular Existence of Mature Odontoblast and Osteoblast Cells in Adult Human Pulp Tissues
A. Eni Juliana,
Z.A. Shahrul Hisham,
S. Nik Marzuki
The dental pulp tissue is essential in dentine development. The existence of Dental Pulp Stem Cells (DPSCs), i.e., osteoblast and odontoblast are to assist in dentine repair and tooth regeneration. The existence of osteoblast that secreted bone matrix directly from pulp tissue has not been reported. The purpose of this study is to determine the existence of odontoblast and osteoblast cells excavated directly from pulp tissues by using molecular markers. The isolated RNA expressing two gene markers, i.e., dentin sialophosphoprotein and osteocalcin which were secreted by odontoblast and osteoblast cells, respectively. The expression of dentin sialophosphoprotein and osteocalcin demonstrated that both odontoblast and osteoblast cells exist in adult human pulp tissues.
Dental pulp tissue located at the centre region of tooth contains soft connective
tissues and cells such as odontoblast, fibroblast as well as defence cells.
This soft and unmineralized connective tissue provides nutrients and sensory
properties to the dentine (Shiba et al., 2003).
The major structure of pulp consists of several layers of odontoblast, cell
free, cell rich and finally loose vascular connective tissue. The large intercellular
space in pulp constitutes of type I and type III collagen fibrils (56 and 41%,
respectively). The dental pulp also contains cells that are responsible for
the formation and turnover of complex non-mineralized extracellular matrices
(Goldberg and Smith, 2004). Most of the pulp cells are
fibroblast-like cells that produce Complex Extracellular Matrices (CEM), which
is substantially different from that of dentin and other soft connective tissues.
Majority of the adult dental pulp contains of macrophages, nerves and capillary
cells (Rodd and Boissonade, 2002). In human, the Dental
Pulp Cells (DPCs) has also heterogenous population that contains progenitor/stem
cells of odontoblast lineage (Alliot-Licht et al.,
2005; Huang et al., 2006). This type of cells
has the ability to proliferate and differentiate into odontoblast under conditions
such as caries and trauma (Murray et al., 2000,
2002). The differentiated odontoblast is believed to
come from a subgroup of precursor cells, i.e., neural-crest derived cells which
reside in the dental pulp.
Odontoblast is highly specialized cell that aligned in a single layer at the
edge of the dental pulp. Therefore, they are the first pulpal cells that are
in contact with dental pathogens (Dommisch et al.,
2005). They play a pivotal role in dental defensive activities during microbial
invasion (Jiang et al., 2006) and also express
β-defensin (Dommisch et al., 2006), Transforming
Growth Factor (TGF)-β (Piattelli et al., 2004)
that induce antimicrobial and anti-inflammatory activities respectively. Odontoblast
cells also produce proinflammatory chemokine interleukin (IL-8) (Huang
et al., 1999) and secrete several collagenous and non-collagenous
proteins to form a unique extracellular matrix. Type I collagen, proteoglycans
and dentin sialophosphoprotein (DSPP) are among the few molecules that are synthesized
and secreted by the odontoblast (Sreenath et al.,
2003). Among these molecules, DSPP has been used as a marker for odontoblast
differentiation and is known as dentin-specific protein (Feng
et al., 1998). On the other hand, osteoblast derived from mesenchymal
stem cells from various development stages of a primitive single cell type (Aubin
and Liu, 1996). They are round cells with an organelle-rich cytoplasm and
play an essential role during bone formation. Mature osteoblast expressed the
extracellular matrix protein, i.e., osteocalcin (OCN), which associated with
the increased of bone matrix mineralization and decreased in osteoblastic cells
proliferation (Candeliere et al., 2001).
In present study, the determination of DSPP and OCN will prove molecularly the existence of odontoblast and osteoblast cells in purified mRNA from human dental pulp. It is also show that cells consist of the common progenitor of odontoblast and osteoblast cells will be differentiated into specific cells upon existence of specific inducer such as differentiated factors or microenvironments.
MATERIALS AND METHODS
Human premolar teeth (18-25 years old) extracted from normal patients were
collected from the Department of Orthodontic, Dental Faculty of UKM, Kuala Lumpur.
The teeth were grooved vertically from the centre of mesial and dental marginal
ridge until cemento enamel junction using dental fissure burs without revealing
the pulp chamber. The extracted teeth were immediately frozen into liquid nitrogen.
The teeth was cut into two-halves and the dental pulp tissues were removed from
the crown and root using dental probe and barbed broach before been homogenized
in TriReagent. The homogenized tissues were allowed to stand for 5 min at room
temperature to ensure complete dissociation of nucleoprotein complexes. Approximately,
0.2 mL chloroform per mL of TriReagent was added into the samples and centrifuged
at 12, 000 g for 15 min at 4°C to separate the mixture into 3 phases: a
red organic phase (containing protein), an interphase (containing DNA) and a
colorless upper aqueous phase (containing RNA).
RNA Isolation of Human Pulp Tissues
The aqueous phase which contains RNA was transferred to a fresh tube
before adding with 0.5 mL of absolute isopropanol per mL of used TriReagent.
The mixtures were allowed to stand for 5-10 min at room temperature and centrifuged
at 12, 000 g for 10 min at 4°C to form a pellet. The RNA pellet was precipitated
with 1 mL of 75% ethanol followed by centrifugation at 7500 g for 5 min at 4°C
before air-drying for approximately 5-10 min. An appropriate volume of either
formamide, DEPC- treated water or 0.5% SDS solution was added to the pellet
and mixed by repeated pipetting before heated at 55-60°C for 10-15 min.
The final preparation of RNA should be free of DNA and proteins, and have an
approximate 260/280 ratio of 1.7. The isolated RNA was used in RT-PCR amplification.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Amplification
One microgram (1 μg) of RNA sample was subjected to RT-PCR amplification
using Access RT-PCR kit (Promega) in a total volume of 20 μL. The sample
was amplified using specific primers (Table 1). The reaction
was incubated in a PCR cycler using programmed cycle indicated in Table
2. The resulting products were separated by 1% agarose gel electrophoresis
and the gels were stained with ethidium bromide, destained in water and photographed
using Alpha Imaging System (Alpha Innotech) under UV light.
||Primer Sequences Used in RT-PCR
|aGAPDH :Glyceraldehydes-3-phosphate dehydrogenase
(NM_002046), bDSPP: dentin sialophosphoprotein (NM_014208), cOCN:
osteocalcin (NM_199173), S: sense and AS: anti-sense. Lower case numbering
at each sense (S) and anti-sense (AS) primer is nucleotide number of designated
||Reverse transcription and PCR cycling profile
|GAPDH: Glyceraldehydes-3-phosphate dehydrogenase (NM_002046),
DSPP: dentin sialophosphoprotein (NM_014208), OCN: Osteocsalcin (NM_199173)
Specific primer based on GenBank nucleotides for human osteocalcin (OCN) was
designed using Primer Premier V5 (Table 1). Primer sequences
for human dentin sialophosphoprotein (DSPP) and glyceraldehydes-3-phosphate
dehydrogenase (GAPDH) were obtained according to Buchaille
et al. (2000) and Papagerakis et al. (2002),
Cloning and Sequencing
The amplification products of GAPDH, OCN and DSPP transcripts were gel-eluted
using Wizard SV Gel and Purification Clean-Up System (Promega) before cloned
into the PCR product cloning vector, i.e., the pGEM-T Easy Vector System (Promega).
The vector was then transformed into JM109 competent cells and the plasmids
constructed for each gene were identified and confirmed by DNA sequencing using
BigDye Terminator sequencing kit (Applied Biosystem). Comparison of cloned human
sequence to known sequence (GenBank/EMBL database) was performed using BLAST
server at NCBI, which gave percentage of identical nucleotide and protein translations.
RNA Isolation from Human Dental Pulp Tissues
The purified total RNA isolated directly from human pulp tissues were taken
from extracted premolar tooth from normal patients. Two bands of RNA, i.e.,
28S and 16S of rRNA which were obtained after the electrophoresis (Fig.
1) showed that RNAs were isolated from the pulp tissues.
Presence of OCN and DSPP Transcripts in Human Pulp Tissues
In order to further demonstrate that the RNA was purified from human pulp
tissues, positive control amplification was performed using a primer set for
glyceraldehydes-3-phosphate dehydrogenase (GAPDH), a house keeping gene (Barber
et al., 2005). The primer resulted in DNA amplification of 195 bp
(Fig. 2a, b; Lane 1). On the other hand,
negative controls were performed using each specific primer set with the RNA
template that was replaced by sterile water which gave no amplification results
(Fig. 2a, b; Lane 3). In order to demonstrate
the activation of DSPP and OCN genes in human pulp tissues, the presence of
their mRNAs were assessed by reverse transcriptase-polymerase chain reaction
||Two types of rRNA (28S and 16S) were directly obtained from
human pulp tissues (lane 1) using TriReagent method and separated by electrophoresis
in 1% agarose gel (DEPC treated) followed by staining with ethidium bromide
||Activation of DSPP, OCN and GAPDH genes in human dental pulp
tissues. RT-PCR was performed on RNAs from pulp tissues with gene specific
primers for DSPP, OCN and GAPDH, which was used as a control; (a) Amplification
of GAPDH (195 bp, Lane 1) and DSPP (293 bp, Lane 2), (b) Amplification of
GAPDH (195 bp, Lane 1) and OCN (270 bp, Lane 2). Negative controls are shown
in lane 3 for both (a) and (b). The PCR products were observed by electrophoresis
on 1% agarose gel and stained with ethidium bromide. 100 bp DNA ladder was
used as marker (M)
||Digestion of plasmid recombinants using Eco RI. The
digestion products were separated by electrophoresis in a 1% agarose gel
and stained with ethidium bromide. Lane 1: 1 kb DNA ladder (Promega), Lane
2: control insert (Promega), Lane 3: GAPDH clone, Lane 4: DSPP clone, lane
5: OCN clone and Lane 6: 100 bp DNA ladder
||Sequence analysis results of OCN, DSPP and GAPDH clones using
DSPP and OCN specific primer sets generated the predicted amplification
products of 293 (Fig. 2a; Lane 2) and 270 bp (Fig.
2b; Lane 2) respectively. The amplification products were cloned into pGEM-T
vector to confirm that the amplicons were the desired products. Those plasmids
which carried the desired genes were digested with a restriction enzyme, i.e.,
Eco RI and resulted in DNA inserts of 195, 293 and 270 bp, respectively
(Fig. 3; Lane 3, 4 and 5).
The amplification products of 270, 195 and 293 bp were successfully cloned
into pGEM-T vector. These clones that carry the desired genes for this study
were further analyzed by sequencing analysis using universal primers, i.e.,
T7 and SP6 as forward and reverse primers respectively. Table
3 shows the sequence analysis results f rom the recombinant plasmids of
195, 293 and 270 bp amplicons. The results showed that the clones were highly
similar with the known sequences obtained from BLASTN analysis.
The aim of this study was to determine molecularly the existence of odontoblast
and osteoblast cells in adult human pulp tissues by the expression of dentin
sialophosphoprotein (DSPP) and osteocalcin (OCN) genes. Present results showed
that DSPP and OCN genes were activated in the mRNA of adult human pulp tissues
when amplified using their specific primers. Most of the previous studies involving
molecular mRNA amplification were performed using dental pulp cells culture
followed by RNAs isolation from the respective culture. Lopez-Cazaux
et al. (2006) isolated total RNA from human pulp cells culture and
assessed the expression of various dentin and bone related transcripts, i.e.,
osteonectin (ON), dentin sialophosphoprotein (DSPP), parathyroid hormone/parathyroid
hormone-related protein-receptor (PTH/PTHrp-R) and glyceraldehydes-3-phosphate
dehydrogenase (GAPDH). However, in this study the RNA was isolated and purified
directly from excavated adult human pulp tissues which were taken from premolar
tooth of normal patients. The isolation and purification of intact RNA was done
by using the modifications of guanidium thiocyanate method which involves extraction
of guanidium thiocyanate homogenate with phenol-chloroform (Chomczynski
and Saachi, 1987). The results from Fig. 1 showed that
the modified method was significantly able to reduce the DNA contamination in
RNA samples. The modification includes incubation of mixture on ice for 5 min
and centrifugation for 15 min. RT-PCR amplification was performed to determine
the involvement of two potential genes, i.e., DSPP and OCN, which were activated
during odontoblast and osteoblast activity, respectively. Amplification of DSPP
and OCN by RT-PCR using their specific primer sets indicated that both genes
were found in adult human pulp tissues hence suggested the existence of mature
odontoblast and osteoblast cells in pulp tissue. In this study, glyceraldehydes-3-phosphate
dehydrogenase (GAPDH), a well-known housekeeping gene was used as our positive
control. GAPDH which is the most common housekeeping gene was also used as comparisons
during gene expression analysis (Barber et al., 2005),
as it is constitutively expressed at the same level in mammalian cells and tissues.
The stimulation of morphologically and functionally odontoblast and osteoblast
cells is crucial in the maintenance of hard tissue specifically in tooth and
bone respectively (Rani and MacDougall, 2000). Osteoblast,
i.e., morphologically as round cells, is responsible for the formation of bone,
by laying down osteoid and mineralizing it. This cell is derived from mesenchymal
stem cells and represents various stages of the development of a primitive single
cell type (Aubin and Liu, 1996). Meanwhile, odontoblast
is the cells responsible for the formation of dentine, the collagen-based mineralized
tissue that forms the bulk of teeth. These cells are post-mitotic neural crest-derived
cells, which are also originated from mesenchymal stem cells. Odontoblast cells
exhibit a tall columnar shape located at the periphery of the dental pulp and
establish a continuous single layer with a clear epithelioid appearance (Arana
Chavez and Massa, 2004). Osteoblast and odontoblast are two cell types which
present in craniofacial complex. They are originated from ectomesenchymal cells
and both of them secrete macromolecules that are necessary for the formation
of dentine and alveolar bone via the respective specific matrix-mediated mechanism.
However, both cells, i.e., osteoblast and odontoblast, synthesizes several common
proteins that involve in calcium and phosphate handling as well as producing
highly similar extracelullar matrices such as type I collagen and one of a major
mineralized matrix protein, i.e., osteonectin.
Dental pulp contains para-axial mesenchyme-derived cells and cranial neural-crest
derived cells (Goldberg and Smith, 2004). These mesenchymal
cells and/or stem cells have been thought to be responsible for hard tissue
formation (Gronthos et al., 2000; Miura
et al., 2003). In in vitro cells culture, these type of cells
that originated from the dental pulp have the ability to differentiate into
odontoblast and osteoblast (Ruch et al., 1995,
Papaccio et al., 2006) by the assistance of a
molecule called Bone Morphogenic Proteins, i.e., BMPs (Nakashima
and Akamine, 2005). BMPs promote the differentiation of mesenchymal cells
into odontoblast and osteoblast by inducing the expression of Runx2 and bone
matrix proteins. In the adult tooth, dental pulp appears to contain progenitors
that can be recruited to differentiate into odontoblast-like cells for tooth
repair (Priam et al., 2005). Papagerakis
et al. (2002) reported that the differentiated odontoblast will produce
DSPP and this protein is not expressed in undifferentiated and primitive pulp
cells. This showed that the mature odontoblast is also present in pulp tissues
as expected since mature odontoblast is important during the formation of dentine.
The DSPP protein is a member of the SIBLING (small integrin binding ligand
N-linked glycoprotein) family, which also includes osteopontin, bone sialoprotein,
dentin matrix protein-1 (DMP1) and matrix extracellular phosphoglycoprotein
(MEPE) (Yunfeng et al., 2005). The gene that
encoded DSPP is located on human chromosome 4q21 and contains multiple phosphorylation
sites. This protein is highly acidic and has an arginine-glycine-aspartate (RGD)
cell attachment domain as it is thought to play an important role in tissue
mineralization (Yunfeng et al., 2005). DSPP is
the initial translational product of DSPP messenger RNA (mRNA) and then cleaved
to dentin phosphoprotein (DPP) and dentin sialoprotein (DSP) (Butler
et al., 2002). DSPP which is expressed by odontoblasts is essential
in dentinogenesis (Zhang et al., 2001; Sreenath
et al., 2003) as it is a dentin-specific protein (Qin
et al., 2002). As a result, DSPP remains to be a significant marker
for odontoblast differentiation.
Osteocalcin (OCN) protein comprises about 2% of the total protein in bone (Knepper-Nikolai
et al., 2002). It is also known as bone Gla protein and is recognized
as a marker for bone formation. It is located on chromosome 1 (1q25-q31) and
is regulated by 1, 25-dihydroxy vitamin D3 (Puchacz
et al., 1989). OCN is a vitamin-K and vitamin-D dependent protein
produced by osteoblasts and is the most abundant. This type of protein is the
non-collagenous protein that is most widely studied in bone. OCN studies in
developing embryos and bone cell models have validated its high specificity
as a marker for the mature osteoblastic phenotype. The osteocalcin gene
is the most thoroughly studied of all bone-specific genes, serving as a model
for regulation by 1, 25 dihydroxy-vitamin D3, glucocorticoids, growth
factors and the transcription factors Runx2/Cbfa1 and Osterix (Knepper-Nikolai
et al., 2002). It is an osteoblast-specific structural gene which
only expressed by fully differentiated osteoblast. Present study had revealed
that the existence of OCN in adult human pulp tissues which were not treated
by any osteoblastic differentiation medium was also expressed. Our results proved
that the undifferentiated and primitive cells in pulp tissue also consist of
matured and committed osteoblast cells.
In conclusion, we have successfully isolated total RNA directly from adult human pulp tissues by using a modified acid guanidium thiocyanate-phenol-chloroform method. This modified method not only reduces DNA contamination in the RNA samples but also generates strong 293 and 270 bp amplicons in RT-PCR. The successful amplification of DSPP and OCN in this study showed that the existence of odontoblastic and osteoblastic cells in undifferentiated and primitive pulp cells population that is in adult human pulp tissues.
The authors would like to thank Ministry of Science, Technology and Innovation for the Fundamental Grant (02-01-02-SF0245), UKM-GUP-BTK-07-15-197, UKM-ST-01-FRGS0020-2006 and UKM-OUP-TKP-18-84/2008.
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