.

1. Absorption Spectrum Analysis of Dentine
Sialophosphoprotein (DSPP) in Orthodontic Patient
Norzaliman, MZ 1,
, Zalhan, MY1
, Farinawati, Y 2
, Asma, A2
, Ken, WSH2
,
Lee, WJ2
, Tan, KF2
, Shahrul, HA3
and Rohaya, MAW 2 a)
1
MIMOS Berhad, Technology Park Malaysia, 57000 Kuala Lumpur.
2
Faculty of Dentistry, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Malaysia.
3
School of Bioscience and Biotechnology, Universiti Kebangsaan Malaysia, 43600 Bangi, Malaysia
a)
Corresponding author: rohaya_megat@ukm.edu.my
Abstract. A force applied during the orthodontic treatment induces inflammation which is essential for tooth movement,
can lead to root resorption. Previous research works have proved a protein biomarker which can monitor the root
resorption during orthodontic tooth movement. Dentine sialophosphoprotein (DSPP) is one of most abundant non-
collagenous protein in dentine. DSPP is released into Gingival crevicular fluid (GCF) during external root resorption. In
this work, we investigate and analysis the absorbance spectrum of human DSPP by using spectroscopy and a qualitative
model using Soft Independent Modelling Class Analogies (SIMCA). The absorption spectrum data will be used as an
indicator for clinical examination of root resorption in orthodontic treatment. The subjects for this study consisted of non-
orthodontic and orthodontic patients based on different clinical treatment period. GCF samples collected from both non-
orthodontic and orthodontic groups showed ultra-violet spectrum range from 244.11 to 259.86 nm. The spectrum data
model accuracy for non-orthodontic and orthodontic patient obtained at 0.91. The result indicates that GCF absorption
spectrums obtained correlated with the duration of orthodontic treatment after the occurrence of Orthodontic-induced
inflammatory root resorption (OIIRR). The qualitative spectrum data model developed is capable to classify samples into
non-orthodontic and orthodontic groups.
INTRODUCTION
Orthodontic-induced inflammatory root resorption (OIIRR) is one of the most common external root resorption
due to orthodontic tooth movement [1]. It is one of the common and undesirable side effects of orthodontic
treatment. OIIRR is a destructive process of the cementum and/ or dentine layers of a tooth due to clastic cell
activity which leads to subsequent loss of root structure of a tooth and affects the longevity of the tooth in severe
cases [2]. Impacted canines, blunted root, long treatment time and usage of heavy force in orthodontic treatment are
factors that can contribute to root resorption [3]. Currently, the detection of root resorption is commonly done by
using panoramic radiograph. However, this conventional way is often associated with limitations of radiation
exposure, technique sensitive, difficulties in standardization, and limited points of view that lead to underestimation
the severity of root resorption [4].
Gingival crevicular fluid (GCF) is the inflammatory transudate that flow out via gingival sulcus. The quantity of
GCF released and also its composition varies depending on the health of the underlying periodontium. GCF is
known to contain an array of biochemical and cellular factors that reflect the health status of underlying

2. periodontium [5]. Dentine sialophosphoprotein (DSPP) is the most abundant non-collagenous protein in dentine [6].
It is critical in the formation of tooth dentine and it is processed by proteases into three major domains: dentine
sialoprotein (DSP), dentine glycoprotein (DGP), and dentine phosphoprotein (DPP). During external root
resorption, DSPP is released into GCF as dentine breakdown products.
Previous research work utilizing optical spectroscopy technique that is proven sensitive to several important
protein biomarkers such as in the study of cancers, including the breast and cervix, and will be the focus of the
subsequent review [7]. The reading absorbance spectrum is obtained when the absorption of light is measured as a
function of its frequency or wavelength [8]. It has been used as the basis for analytical procedures in various fields,
including healthcare, where together with other techniques it assists in developing methods for diagnosing disease
[9]. Analysis of dentine proteins in GCF is a potentially safer method of quantifying root resorption compared with
conventional radiographic methods. The aim of the present study is to investigate the absorbance spectrum of
orthodontic patient via DSPP biomarker by using absorption spectroscopy method and to analyze the spectrum
based on a qualitative model. The model is built using Soft Independent Modelling Class Analogies (SIMCA)
algorithm. SIMCA is a supervised pattern recognition that based on disjoint principal component analysis (PCA) for
each classification [10]. The absorption spectrum data will be used to develop a prototype for clinical examination
of root resorption orthodontic treatment which is quick and easy to use, no drawbacks of radiation exposure, less
time consuming and inexpensive.
MATERIALS AND METHODS
Subject selection
Prior the commencing of the subject recruitments, approval for the study was obtained from the Research Ethics
Committee, The National University of Malaysia (Ethical approval number, UKM PPI/111/8/JEP-2018-438). The
subjects for this study consisted of 7 orthodontic patients which were further categorized into 2 samples of three-
months into treatment (T3), 2 samples of six-months into treatment (T6), and 3 samples of twelve-month into
treatment (T12). For non-orthodontic patients (control), 3 samples were recruited. Orthodontic patients were
consecutively sampled using inclusion criteria as follows: Healthy subjects without any systemic disease(s); not on
any medications; wearing fixed appliances; having full permanent dentition; no history of trauma to their teeth. The
inclusion criteria for non-orthodontic patients were similar to the orthodontic group except that they did not receive
any orthodontic treatment (T0).
GCF sampling
Informed consent was obtained from the patient or the parent or guardian prior to the commencement. Optimal
oral health was achieved in all subjects prior to the study where any supragingival plaque was removed from
sampling sites. Basic oral examination was carried out to ensure optimal oral hygiene and gingival status as the
concentration of total protein in gingival crevicular fluid (GCF) has significant correlation with gingival index,
pocket depth measurements, where it reflects the clinical status of gingival and periodontal tissues [11]. Central
incisor of patient was isolated using cotton rolls and saliva ejector to remove the remaining saliva to prevent
contamination of saliva which consists of various type proteins to improve the accuracy of the absorbance spectrum
readings. Gingival fluid collection strips (Periopaper, Oraflow, Smithtown, N.Y.) were placed within the sulcus
(intrasulcular method) approximately 1 to 2 mm into the gingival sulcus and left in situ for 60 seconds [12]. The
fluid seeping out was absorbed by the strips. GCF was sampled from the mesial and distal gingival crevicular
margins of the central incisor. Every collection of samples was taken 3 times at the same site with one-minute
resting intervals to permit the replenishment of GCF into the gingival sulcus. The undipped part of periopaper with
GCF was cut and removed using scissors. Then, all 6 dipped strips were placed immediately into 1.5 mL
microcentrifuge tube containing 300μL of protease inhibitor (Cocktail Kit, MP Biomedicals, LLC, US). The dipped
paper strip with GCF was eluted by centrifugal filtration at 400xg for 10 minutes at 4o
C with centrifuge machine
(Heraeus Fresco 21 centrifuge, Thermo Scientific). The samples were stored at -80o
C.

3. Absorption Spectroscopy Characterization
The spectroscopy experimental setup or specifically the reflective absorption spectroscopy optical setup used in
this work shown in Figure 2, consist of a light source, an optical probe, a special cuvette permeable ultraviolet
spectrum, a cuvette holder and a highly sensitive spectrometer. The light source is utilizing a deuterium lamp which
is able to generate an ultraviolet spectrum from 180nm onward until 400nm. The ultraviolet spectrum result
displayed by the special software (Spectrasuite) which comes with the spectrometer. The centrifugated GCF samples
were pipetted into a special quartz glass cuvette (Binful, China) with dimension of 12.5mm x 12.5mm x 45mm and
able to hold maximum volume of 350μL aqueous GCF sample. The cuvette allows ultraviolet to near infrared
spectrum, 200nm to 2500nm wavelength pass through the sample. However, the absorption spectroscopy method
used in this experiment aims to probe DSPP in the GCF sample. Hence, the cuvette contains only GCF and protease
inhibitor is used as a baseline or as a reference sample, where no DSPP protein is presence. A bifurcated optical
probe is used as a waveguide to guide the light beam from the light source onto the sample and collect back the
reflected light beam from the sample to the spectrometer. The end of optical fiber probe and the quartz cuvette are
placed in a custom cuvette holder to avoid unnecessary movement of the said components during the
characterization measurement session. The cuvette was reused by pipetting out the old sample followed by multiple
rinsing with distilled water and airdrying of the cuvette’s inner wall, then filled with new sample. The absorbance
values of the samples were recorded. A graph of absorbance spectrums for every samples were made and the most
representative absorbance results from each sample groups were chosen and reconstructed to another graph.
FIGURE 2: Optical setup for sample characterization using absorption spectroscopy
RESULT AND DISCUSSION
Spectroscopic measurements are very sensitive and non-destructive, and require only very small amounts of
material for analysis [8]. The absorption intensity of the sample calculated accordance to Lambert Law,
− log 𝑇 = єbc (1)
where the transmittance, T is the ratio of light intensity passed through the sample, after and before. The
transmittance is equals to the product of the molar absorbtivity of target protein (є), the light path of the sample (b),
and the concentration of the compound in the solution (c). Since the the molar absorbtivity of target protein and the
light path of the measured sample in the cuvette remain unchanged, therefore the absorption of the DSPP in the GCF
sample depends linearly on its concentration. Gingival crevicular fluid (GCF) samples from both groups

4. (orthodontic and non-orthodontic patients) showed absorbance spectrum in the range of wavelength from 244.11 to
259.86 nm. However, all GCF samples showed different intensity levels of absorbance spectrum. GCF samples in
the non-orthodontic patient group (T0) showed lower peak of absorbance spectrum compared to the GCF samples
from the T3, T6 and T12 orthodontic groups as shown in Figure 3. The mean values of the GCF samples shows that
the mean absorption spectrums of the samples are higher in intensity when the duration of treatment is longer as
shown in Table 1. The most representative results from T0, T3, T6 and T12 group shows in Figure 3 indicates that
the longer the orthodontic treatment duration, higher absorption spectrums were noted. The absorbance spectrums of
T0, T3, T6 and T12 were proportional to the duration of treatment.
Months N Wavelength peak (nm) Absorbance intensity (Arbitrary)
T0 3 244.11 50.00
T3 2 253.07 84.50
T6 2 265.22 107.00
T12 3 259.86 191.67
TABLE 1: Mean values of absorbance spectrum and wavelength peak of T0, T3, T6 and T12
FIGURE 3: Absorbance spectrum of representative samples from the mean value of T0,T3, T6, and T12
In our study, the GCF sample was collected from the maxillary central incisors of patients according to the
selection criteria. The maxillary central incisor was selected as it is the most accessible tooth for collection of GCF.
It also has the highest percentage of severe root resorption due to orthodontic tooth movement [3]. The subjects
selected did not experience any history of dental trauma as dental trauma may induce inflammation and root

5. resorption [13]. The duration of force application or active treatment is also one of the risk factors related to
orthodontic treatment as well as increased levels of apical root resorption [3].
From Table 1, GCF samples from both groups (orthodontic and non-orthodontic patients) showed absorbance
spectrum in the range of wavelength from 244.11 to 259.86 nm. However, all GCF samples showed different
intensity levels of absorbance spectrum. Most of the GCF samples in the non-orthodontic patient group (T0) showed
lower peak of absorbance spectrum compared to the GCF samples from the T3, T6 and T12 orthodontic groups as
shown in Figure 3. The most representative results from T0, T3, T6 and T12 group indicates that the longer the
orthodontic treatment duration, higher absorption spectrums were noted. This shows that in orthodontic groups (T3,
T6 and T12), the protein content in the GCF samples was increased. Thus, this could be an indicator that higher
amount of DSPP was released into GCF at least 3 months after orthodontic treatment. This finding supported by
Kereshanan who had successfully shown an increase in DSP levels at 3 months following the start of orthodontic
treatment [14]. Proteins absorb ultraviolet light with absorbance maxima at 300 nm and 200 nm. Amino acids with
aromatic rings are the primary reason for the absorbance peak at 290-300 nm. Peptide bonds are primarily
responsible for the peak at 200 nm. Secondary, tertiary, and quaternary structure all affect absorbance spectrum.
Moreover, factors such as pH, ionic strength, sample concentration can alter the absorbance spectrum. Therefore,
different proteins can have different absorption coefficients and even the wavelength spectrum. The fact that
increased orthodontic treatment time is associated with a higher risk of root resorption and this is also reflected by
the higher levels of DSPP as time increases. Thus, the findings indicate that the increasing absorbance spectrums
from T3 to T12, was due to increased releasing of DSPP into GCF after the occurrence of OIIRR.
Further analyze the spectrum data, a qualitative model is build using SIMCA. The goal of SIMCA is to obtain a
classification rule for a set of known groups thus it is used to distinguish non-orthodontic and orthodontic where the
similarity within a class is emphasized [15]. In this analysis, the SIMCA algorithm was performed using
MatlabR2013a software and all the result depicted in Table 2 below.
Actual classes
Non-ortho Ortho
Predicted
classes
Non-ortho 3.0 0.0
Ortho 1.0 7.0
(a)
Non-ortho Ortho
True Positive (TP) 3 7
False Positive (FP) 0 1
True Negative (TN) 1 0
False Negative (FN) 7 3
(b)
Parameter Value
Non-Orthodontic Orthodontic
Sensitivity 1.00 0.88
Specificity 0.75 1.00
Precision 1.00 0.75
Accuracy 0.91
(c)
TABLE 2: (a) The prediction result of classification of non-orthodontic and orthodontic patient. (b) Statistical parameters of
classification analysis. (c) Model sensitivity, specificity, precision and accuracy calculated value.
Detecting non-orthodontic samples as non-orthodontic is known as true positive (TP) case while false negative
(FN) is non-orthodontic samples incorrectly identified as orthodontic. True negative (TN) is the case where
orthodontic samples are correctly classified as orthodontic whereas false positive (FP) when orthodontic samples are
identified as non-orthodontic. Only one sample of non-orthodontic sample predicted as orthodontic sample while all
orthodontic samples predicted as orthodontic according to the matrix table shown in Table 2. Model accuracy was
calculated by the number of correctly classified for non-orthodontic and orthodontic divided by whole data.
The effect of OIIRR is inevitable but its progression can be prevented by early detection. Hopefully, the
usage of potential biomarkers for detection root resorption is useful not only in the early intervention for root
resorption, it can also be used as a chairside tool to inform patient about the risk of root resorption prior to treatment
Furthermore, it also can reduce the repeated radiographs exposure in monitoring root resorption.

6. CONCLUSIONS
The result outcome has demonstrated the potential absorption ultra-violet spectroscopy method as an analytical
method in detecting DSPP in GCF which is a biomarker for root resorption. The result highlights the absorbance
ultra-violet spectrum of DSPP in GCF at the wavelength from 250 to 300 nm with qualitative spectrum data model
accuracy at 0.91. The results of this study indicate that the spectrum absorbance of DSPP in GCF from
orthodontically treated patients is higher than the non-orthodontically treated patients.
ACKNOWLEDGEMENTS
The authors would like to acknowledge with gratitude the research team and laboratory technicians of Photonics
Department Laboratory, MIMOS Berhad and Faculty of Dentistry, National University of Malaysia (UKM) for their
cooperation and support. This research paper was funded by University Kebangsaan Malaysia (UKM Grant Code:
GUP-2017-002).
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