Journal club about effect of diabetes on elemental levels of dentine

1. EFFECT OF DIABETIS ON
ELEMENTAL LEVELS AND
NANOSTRUCTURE OF ROOT
CANALDENTINE

2. The Journal of Endodontics, the official
journal of the American Association of
Endodontists, publishes scientific
articles, case reports and comparison
studies evaluating materials and
methods of pulp
conservation and endodontic
treatment.
Impact factor-4.422

3. INTRODUCTION
Dentin is a highly mineralized tissue that constitutes most of a tooth's
structure.
Healthy dentin has long and thin tubules, surrounded by inorganic
peritubular dentin with more than 90% mineral content.
At the nanoscale level, dentin displays a natural exchange of trace
elements that are critical for its mechanical and crystalline characteristics.
Nanometer-sized hydroxyapatite crystals in the intertubular collagen
matrix can readily accommodate trace mineral substitutions, resulting in
changes to the mechanical, physical, and chemical behavior of dentin .

4. Diabetes mellitus (DM) is a disease of the endocrine system that impairs
the regulation of blood glucose levels and disrupts mineralization by
interfering with its production
Its prevalence has been rapidly increasing, affecting 37.3 million
Americans in 2019, of whom 90% were diagnosed with type 2 DM.
Diabetes has systemic effects on all organs and tissues in the body,
including those in the oral
cavity.
However, studies on the level of trace elements in diabetic teeth are
sparse

5.  Patients with diabetes have higher rates of root canal failure, pulpal
infections, pulp sensitivity, and decreased retention of endodontically
treated teeth", which could be linked to trace minerai imbalances.
 Deficiencies in essential trace elements magnesium, Iithium, manganese,
strontium , zinc , and selenium can affect dentinal mechanical properties
resulting in reduced peritubular dentin decreased microhardness, reduced
shear bond strength lower root fracture resistance, increased tubular
density and increased susceptibility to poor dentin instrumentation.
 Diabetes-related dentin softness can damage root integrity and result in
endodontic procedural errors, such as apical transportation, ledge
formation, and strip perforation, potentially caused by a decrease in the
quality and size of dentin apatite crystals

6.  Dentin's microstructure is typically studied using x-ray techniques,
scanning electron microscopy (SEM), and transmission electron
microscopy (TEM) at the micrometer to upper nanometer scale.
 However, few studies have explored its nanostructural characteristics, and
no study has investigated how dibetes affects it, although the condition
interferes with the natural mineralization process ,leading to changes in
root canal dentin's mechanical properties that may cause higher rates of
endodontic procedural failures.

7.  Therefore, this study aimed to compare the nanostructural characteristics
and level of inorganic trace minerals in root canal dentin in healthy and
diabetic patients using inductively coupled plasma mass spectrometry (CP-
MS) to analyze mineral content and high-resolution TEM (HRTEM) to
evaluate the shape and quantity of nanoscale hydroxyapatite crystals.
 The first null hypothesis was that there was no difference served in the
level of trace element in the dentin between diabetic and nondiabetic
specimens. The second null hypothesis was that there were no differences
in the nanostructure of dentin between diabetic and nondiabetic
specimens

8. MATERIALS AND
METHODS
TEETH COLLECTION
 The study protocol was approved by the institutional review board (#Pro2019002923) at
Rutgers School of Dental Medicine.
 Twenty first premolars were selected from diabetic and nondiabetic patients who had
teeth extracted for orthodontic purposes.
 The inclusion criterion for the diabetic group was type 2 diabetes donors with no other
systemic diseases (verbaly declared by the donors) who have been diabetic for 5 to 15
years (glycemic control: HbA1C-6%-0.5%). We had similar inclusion criteria with the
nondiabetic patients, who also must not have had any systemic disease history.
 Exclusion criteria were dentine donors younger than 20 and older than 60 years, smoking
history or parafunctional habits; low fat, vegetarian diet: pregnant or lactating, teeth with
any cracks, defects, or cervical caries (confirmed using stereomicroscope) teeth with
previous root canal procedures; and teeth stored in antibacterial or fixative solutions
 .To disinfect the teeth, they were stored in 0.5% chloramine T at 4°C for up to 15 days
before use , followed by storage in distilled water until tested.

9. SPECIMEN SIZE SELECTION
G-Power software (University of Cusseldort, Dusseldorf, Germany) was used
to determine the specimen size. Considering an alpha-type error of 0.05 and
a power of 90% (effect size 0.8), 10 specimens were estimated for each
group for both ICP-MS and HRTEM

10. SPECIMEN PREPARATION
 The premolars were decoronated under water using a low-speed Isomet
diamond saw (Buebler, Lake Bluff, NY), and 40 dentin discs (2 mm thick)
were obtained from the coronal third of the root portion (immedarely
adjacent to the pulp chamoer), similar to our prior studies. Each tooth
provided 2 dentin discs (1 disc for ICP-MS and disc for HATEM The dentin
specimers were then wet-ground with a 320-grit and polished with a 600-
grit to obtain a standardized flat surface:

11. CHEMICAL CHARACTERIZATION.
 Each dentin disc was ground using a smart dentin grinder (Komistaßio, Fort
Lee, NJ) and weighed (0.2 g) on a precision balance.
 The specimens were then digested with 5 ml, al 86% nitric acid, 4 ml of
Mill-Q water, and 2 ml of 30% hydrogen peroxide, and the resulting
solution was diluted with distilled water and analyzed with ICP-MS .
 Calibration of ICP-MS was performed using a 1000 mg/L multielement
calibration standard solution of the analyzed elements (Mg. Li, Cu, Mn, Sr.
Zn, and Se .
 All measurements were conducted in triplicate, and detection limits were
calculated as 3 times the standard deviation of the blank values

12. APATITE CRYSTAL CHARACTERIZATION
 Dentin specimens were pre-thinned to 50 to60 micrometer using a smart dentin grinder and
then dispersed onto a 200-mesh copper TEM grid that had been coated with a thin carbon film.
 To ensure sufficient electron transparency, nanoscale dentin specimens (100 nm) were
prepared by using a focused ion beam operated with a liquid gallium ion source at a voltage of
30 kV and a current of 50 pA.
 Subsequently, the diabetic and nondabetic specimens were imaged with an HRTEM operated
at 200 kV. TEM micrographs at x100.000 magnification were used to determine morphological
and crystallographic differences between diabetic and nondiabetic groups by qualitatively
recording crystal shape and quantitatively measuring the number of crystals in 2500 nm area
using a modified Image J protocols.
 Each micrograph was opened of the Images J and the scale of the image was set by magnifying
the image and drawing a straight Iine from one end of the scale maker to the other.
 The bandpass filter and contrast adjusted to separate definitive Iines .
 Finally freehand selection tool was used to trace each crystal and provide surface area
measurements.

13. STATISTICAL ANALYSIS
 The statistical analyses were performed SPSS 25.0 .
 Normal distribution of the data was confirmed by the Kolmogorov-
Smirnov test.
 Intergroup comparisons of the means for elemental and crystalite levels
were analyzed using the Student t test.

14. RESULTS
 A total of 7 trace elements (Cu, Li Zn, Mg, Mn, Se, and Sr) were
analyzed in both dabetic ad nondiabetic dentin specimens, all of
which were found to be significantly altered in the diabetic
specimens .
 The concentrations of Li , Mg , Mn ,Se were significantly lower n
diabetic specimens than in nondiabetic specimens, while the
concentration of Cu ppm was significantly higher in diabetic
specimens than in nondiabetic specimens .

15.  HRTEM provided 3-dimensional structural information, revealing that
diabetic
 Dentin had significantly more apatite crystals in the 2500 m² area
compared with nondiabetic dentin.
 Diabetic dentine exhibited a less compact structure with smaller crystalites
and some porosties.
 Both diabetic and nondiabetic dentin had needle-like mineral crystals with
no observable qualitative differences morphology.
 However, the crystaline region in diabetic dentin exhibited more
polycrystalinty, with some amorphous region detected

16. DISCUSSION
 This study investigated the level of inorganic trace elements and nanostructural
characteristics of root canal dentine in healthy and diabetic patients using a
combination of chemical analysis with ICP-MS and ultrastructural characterization with
HRTEM.
 There are several inorganic trace elements involved in ionic substitutions into the
dentinal hydroxyapatite lattice, such as Cu, Li, Zn. Se, Sr. Mn, and Mg. These elements
were selected because of their integral roles in dentin apatite and their known
association with diabetes related effects on dentine.
 Coronal dentin was analyzed in this study because of its proximity to the pulp
chamber, which houses a high density of blood vessels that facilitate the exchange of
trace elements in dentin hydroxyapatite,.
 Although our investigation specifically targeted coronal dentin, it is important to note
that dentin is a highly mineralized tissue, and as such, ultrastructural differences may
also exist in radicular dentin.
 Additional studies focusing on different regions along the root are needed to
adequately understand the varations in trace elements and nanostructure throughout
the entire tooth structure.

17.  ICP-MS was used in this study because of its prevalence in accurately
measuring trace elements with high sensitivity, particularly in small sample
sizes .
 SEM-energy dispersive spectroscopy is another available method for
confirming elemental information but requires a higher voltage and
current to generate sufficient x-ray signals, leading to charging issues on
nonconductive samples such as dentin
 ICP-MS analysis of dentin specimens showed significant changes in trace
elements (Cu Li, Zn, Mg, Mn. Se, and Sr) in diabetic patients compared
with healthy patients .
 Diabetic dentin specimens exhibited lower levels of Li, Zn, Mg, Mn, Se,
and Sr, and higher levels of Cu
 The altered levels of inorganic trace minerals in diabetic dentin can be
explained by peritubular demineralization and altered pulp calclication
resulting from larger dentinal tubules and higher tubular density.
 Patients with diabetes generally exhibit larger tubules and denser
capilares, leading to increased bacterial colonization within the tubules.

18.  The presence of altered pulpo calcification may serve as a response to
bacterial invasion through the dentinal tubules
 This calcification process can result in pulp chamber narrowing, reduced
pulp volume, and potential impairment of pulo function, ultimately
affecting the diffusion of trace elements and contributing to peritubular
demineralization
 Decreased levers of mineral ions, including Li, Mg, Zn, and Sr. in diabetic
patients can damage the cells in the dentin-pulp complex, as these
minerals are important for dentin mineralzation and pup vitality.
 Diabetes also disrupts the dental pulp by altering circadian rhythms,
inhibiting dentinal bridge formation, and causing pathological
calcifications and pulp thickening.
 This reduced pulp vitality may compromise the absorption and release of
inorganic trace elements essential for dentin function.

19.  During dentin biomineralization, inorganic nanocrystals (Mg, Zn, Sr. L. Mn,
Se etc.) precipitate within organic matrices. Se substituted dentin
hydroxyapatite has been found to effectively prevent secondary infections
after root canal procedures by penetrating deep into dentinal tubules"
However, interference of DM with Se
 between Se and phosphate ions which could decrease the antibacterial
properties of dentin apatite. In addition, low levels of Mn in patients with
diabetes can adversely affect dentin's structural properties and its
response to dental treatments. Elevated Cu vels in diabetic dentin could be
attributed to increased protein glycation, resulting in a more fragile
material and impaired dentinal mechanical properties".

20.  Differences in the concentrations of specific ions may affect dentin
mineralization and crystal quality
 This may involve adjusting treatment protocols, such as using different
filling materials or root canal irrigants, to account for the altered
mechanical properties of diabetic dentin caused by reduced mineral
content.
 DM also interferes with the quantity and quality of nanocrystals, which
may influence the mechanical properties of dentin.
 HRTEM was used in this study to provide 3-dimensional structural
information on diabetic and nondiabetic dentin at a near-atomic level, a
relationship that cannot be determined through SEM".
 The present work used a modified Image J protocol to provide surface
area measurements, as the threshold function posed a challenge due to
the crystals being clustered together and overlapping.
 Quantitative analysis confirmed significant differences in the number of
apatite crystals within 2500 nm² (P<051. with diabetic specimens having
approximately 2.5 times the average number of crystalites observed in
nondiabetic specimens.

21.  . The relatively small size of the observed crystalites suggests some degree
of demineralization in diabetic dentin", which may be caused by
deficiencies in minerals from the pulpal blood supply and reduced
expression of alkaline phosphatase.
 DM can have adverse effects on dentin formation in the early growth stage
and affects odontoblasts similarly to now is affects bone formation .
 These metabolic changes in odontoblasts could also explain the inhibitory
effects of hyperglycemia on dentin mineralization, leading to a weaker
tooth structure that may be more prone to endodontic procedural tailures
and overinstrumentation


22.  The mechanical properties of teeth depend not only on their composition
but also their mineral nanostructure.
 HRTEM revealed a significant difference in the nanostructure of debetic
and nondiabetic dentin.
 Diabetic dentin exhibited polycrystalline grans whereas healthy dentin
exhibited well organized and tightly packed needle-like apatite crystalites,
which may contribute to the strength of teeth.
 This difference in nanostructure indicates that the mechanism of
hydroxyapatite formation differs due to varying levels of trace elements in
diabetic and nondiabetic dentine.

23.  Ortiz-Ruiz et also studied differerices between crystalline nanostructures
in dentin and enamel hard tissues and showed that the crystalograffiac
characteristics might determine the mechanical properties of these
tissues.
 For instance, resistance to fracture depends on the hardness and density
of its crystal components, the size, the degree of crystallinity, and the
orientation of the crystal.
 In the case of diabetic patients endodontically treated teeth are more
prone to root fracture, potentially due to difference in mineral
nanostructures when compared with nondiabetic specimens.
 More specificaly, the lower fracture resistance of diabetic specimens might
be due to decreased diffusion of trace elements nom the dentral pulp or
the exposed root surface in the coronal third

24.  Specimens from type 2 dibete patents with variaing durations of diabetes
were used in this study.
 However the heterogenicityof type 2 diabetes development and
management could have potentially influenced the results.
 The wide range of patients apes (20 to 60 years) within a small chart in 10
per group) may also influence the measures of trace elements quantified
and affect the generalizability of the findings.
 In addition, this study did not consider sex differences as a biological
variable
 Future studies should expand to type 1 diabetes with larger cohorts and
better control for variabies such as age, duration of disease, and sex to
provide a more comprehensive understanding of the effects of diabetes on
dentin structure.

25.  Mechanisms behind these changes are not yet fully understood, and
appear to vary depending on the mineral involved.
 Future x ray diffraction studies may clarify how the crystal's shape, size,
crystallinity, and orientation are modified by inorganic trace: mineral
concentration. Appropriate
 Knowledge regarding this may aid in the development of biomatestals that
can , prometo the preferred substitution and orientation of hydroxyapatite
crystals to strengthen dentin for better root canal outcomes in diabetic
patients.

26. CONCLUSION
 The study's findings suggest that there are ultrastructural differences in
the dentin of diabetic patients compared with healthy patients, as
observed through changes in trace element levels, crystalite quantity, and
crystallite quality.
 These differences may have implications for root canal treatment,
potentially resulting in increased procedural errors and decreased
retention of endodontically treated teeth in patients with diabetes