Original Article

Comparative evaluation of the effect of two natural

collagen cross‑linkers on microtensile bond
strength of self‑etch adhesive system to dentin after
contamination with blood and hemostatic agent: An
in vitro study
Amarjot Kaur D S Manihani, Sanjyot Mulay, Lotika Beri, Anita Tandale, Abhilasha Bhawalkar, Raj Dalsania1
Department of Conservative Dentistry and Endodontics, Dr. D. Y. Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Pune,
Maharashtra, 1Department of Pediatric and Preventive Dentistry, Pacific Dental College, Udaipur, Rajasthan, India

Abstract
Background: Cavity preparation often causes gingival bleeding which can be controlled by hemostatic agents (HAs). These
along with blood act as contaminants and hamper the bonding mechanism. Collagen cross‑linkers (CCLs) are agents known
to increase the bond strength (BS) to dentin. Hence, the purpose of this in vitro study was to determine the effect of two different
CCLs, proanthocyanidin (grape seed extract [GSE]) and hesperidin on the microtensile BS (µTBS) of a self‑etch adhesive (SEA)
system to dentin which was contaminated with blood and a HA.
Materials and Methods: Thirty‑six extracted human molars were collected, and their occlusal surfaces were sectioned to expose
the dentin. The teeth were randomly divided into four groups: Group I – Control, Group II – Contamination with blood and
HA, Group III – Application of GSE after contamination, and Group IV – Application of hesperidin extract after contamination.
The SEA was applied, followed by the use of a nanocomposite. Dentin‑composite rods were obtained from each group, and
µTBS testing was done. The fracture pattern was visually classified as an adhesive failure at the interface, cohesive failure in
composite, or dentin. The scanning electron microscope (SEM) analysis was done for two samples from each group. Statistical
analysis was done using the Student’s unpaired “t” and ANOVA test.
Results: Group II showed a statistically significant reduction in µTBS in comparison to Group I. This was overcome in Groups III
and IV. Hesperidin showed marginally better results than GSE.
Conclusions: The use of GSE and hesperidin increases the µTBS of composite resin to dentin postcontamination with blood
and ViscoStat Clear with Single Bond Universal Adhesive.
Keywords: Blood contamination; collagen; cross‑linkers; dentine; grape seed extract; hemostatic agent; hesperidin;
microtensile bond strength; proanthocyanidins; self‑etching adhesive

INTRODUCTION
Ongoing trends in adhesive dentistry emphasize the evolution
Address for correspondence: of bond strengths (BSs) to obtain durable restorations. Self‑etch
Dr. Amarjot Kaur D S Manihani,
No. 601/A Wing, Dheeraj Residency, Goregaon West, bonding agents are popularly employed as they consume
Mumbai ‑ 400 104, Maharashtra, India.
E‑mail: amarjot.kaur04@gmail.com This is an open access journal, and articles are distributed under the terms
of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0
Date of submission : 09.05.2023 License, which allows others to remix, tweak, and build upon the work
Review completed : 01.06.2023 non‑commercially, as long as appropriate credit is given and the new
Date of acceptance : 05.06.2023
creations are licensed under the identical terms.
Published : 28.07.2023
For reprints contact: WKHLRPMedknow_reprints@wolterskluwer.com
Access this article online
Quick Response Code: How to cite this article: Manihani AK, Mulay S, Beri L,
Website:
https://journals.lww.com/jcde Tandale A, Bhawalkar A, Dalsania R. Comparative evaluation of
the effect of two natural collagen cross‑linkers on microtensile
bond strength of self‑etch adhesive system to dentin after
DOI:
10.4103/jcd.jcd_312_23
contamination with blood and hemostatic agent: An in vitro study.
J Conserv Dent Endod 2023;26:466-71.

466 © 2023 Journal of Conservative Dentistry and Endodontics | Published by Wolters Kluwer - Medknow
 Manihani, et al.: Effect of collagen cross‑linkers on microtensile bond strength after contamination
less time, require no rinsing, and cause less sensitivity.[1]
Nevertheless, they face the challenge of bonding to variable
tissues. In the clinical scenario, an ideal contaminant‑free
substrate is necessitated for an optimal bonding procedure.
Tooth cutting commonly leads to gingival bleeding which
might be due to iatrogenic tissue injury/gingivitis. This may
be seen in deep class II caries where there is a subgingival
extension.[2] Hence, hemostatic agents (HAs) are commonly
used for a blood‑free operating field.
HAs are acidic solutions. Their pH ranges from 0.7 to 2.0,[3]
and they are applied for a stipulated time and rinsed off.
Research suggests that self‑etch adhesives (SEAs) fail to
eliminate these contaminants completely. This is due to their
short resin tag formation and a comparatively thin hybrid
zone. Moreover, dentin matrix metalloproteinases (MMPs)
are activated due to the acidic nature of adhesives, resin
cements, and HAs,[3,4] which results in hydrolytic destruction
of resin and collagen.[5] This is seen in the dentin matrix
where the resin penetration is incomplete and is one of the
primary features of failed restorations.
Dentin is majorly composed of Type 1 collagen. Hence, the
current concepts to enhance bonding incorporate agents
that improve the nature of collagen. This is primarily done
Figure 1: Flowchart depicting the sequence of events
Journal of Conservative Dentistry and Endodontics | Volume 26 | Issue 4 | July-August 2023
by increasing the inter and intramolecular cross‑links.
Application of collagen cross linkers (CCLs) on dentin
before bonding aids to establish this scenario.[6]
Proanthocyanidins (PAs) are plant origin polyphenols
that are naturally occurring CCLs, for example, grape
seed extract (GSE).[7,8] Previous studies state that their
application increases the BS of composite to dentin.[6,9]
Hesperidin, another naturally occurring CCL, has also shown
increased BS. Moreover, it also has potent antioxidant and
anti‑inflammatory properties.[10]
Although the effect of these CCLs has already proven to be
beneficial to the composite‑dentin BS, no study has been
carried out to evaluate their effect after dentin contamination.
Hence, the purpose of this in vitro study was to evaluate how
two different CCLs affected the microtensile BS (µTBS) of a
SEA to dentin postcontamination with blood and a HA.
MATERIALS AND METHODS
Thirty‑six extracted permanent human molars were
collected, and their occlusal surfaces were sectioned to
expose the dentin. The teeth were randomly divided into
four groups [Figure 1].
467
Manihani, et al.: Effect of collagen cross‑linkers on microtensile bond strength after contamination

• Group I: Control group. No contamination of the teeth.
• Group II: Blood + ViscoStat Clear. Uncoagulated human
blood was applied to the dentin of each tooth and kept
for a minute. It was rinsed with water for a minute and
then blotted with absorbent paper. Then, HA, ViscoStat
Clear (Ultradent Products, Inc.) application was done
for a minute. This was followed by rinsing for a minute
and blot drying.
• Group III: Blood + ViscoStat Clear + 6.5% GSE. The
steps in Group II were followed by application of 6.5%
GSE, i.e. PA for 5 min and rinse with water and blot
drying (a quantity of 6.5 g of GSE in the form of powder
was collected from the capsules Zenith Nutrition,
Medizen Labs Pvt. Ltd., India, and dissolved in 100 mL
of distilled water to obtain 6.5% GSE).
• Group IV: Blood + ViscoStat Clear + 10% hesperidin.
The steps in group II were followed by the application
of 10% hesperidin (10 gm of hesperidin powder in 100
mL of distilled water (Sigma-Aldrich) for 5 min, a water
rinse, and blot drying).
Application of the SEA, Single Bond Universal also known
as Scotchbond™ Universal (3M ESPE St. Paul, MN, USA)
was done on all teeth following the manufacturer’s
instructions. A nanocomposite, Filtek™ Z350 XT composite
(3M ESPE, St. Paul, USA) was applied on the bonded
surfaces incrementally. The total height was 8 mm to allow
gripping during the tensile testing. Increment thickness
was limited to 2 mm, and curing was accomplished for 40 s
per increment and cured in four increments to form a core
8 mm high.
Samples were kept for 24 h in distilled water. They were
sectioned longitudinally to procure 72 rods (18 rods in each
group). The dimension of each rod was 1.6 cm long and
1 mm × 1 mm thick, with the dentin–composite interface
in the middle. All the samples were then subjected to µTBS
loading.
Scanning electron microscope analysis
The fracture pattern was visually classified as follows:‑
1. Adhesive failure at the interface
2. Cohesive failure in dentin
3. Cohesive failure in composite.
Two samples were randomly chosen from each group and
scanning electron microscope (SEM) analysis (2500X) was
done to assess the micromorphology of the fractured
interface.
RESULTS
Statistical analysis was done by the comparison of the
mean and standard deviation values of µTBS (MPa) among
uncontaminated (control), blood + HA, GSE, and hesperidin
groups [Table 1].
The Student’s unpaired “t” test was applied. The result
of the study revealed that the highest mean µTBS values
were for Group I (31.84 ± 0.27 MPa), followed by Group IV
(31.23 ± 0.79 MPa) and Group III (30.45 ± 0.92 MPa). Group II
had the least values (15.62 ± 0.98 MPa) [Graph 1].
One way ANOVA with Tukey–Kramer multiple comparison
test was carried out at a 5% level of confidence. Intergroup
comparison between groups showed a statistically
significant difference in µTBS (P < 0.05). Groups I and II (P
= 0.0001), I and III (P = 0.0234), II and III (P = 0.0001), and
II and IV (P = 0.0001). However, the comparison showed
statistically nonsignificant results between Groups I and IV
(P = 0.0951) and Groups III and IV (P = 0. 9948).

Microtensile bond testing

• After aligning the jaws of the universal testing machine
in a straight line, the specimens were attached to a
jig (fixtures) with cyanoacrylate adhesive
• Tensile stress application (1 mm/min) until
specimen fracture was done. This load obtained in
Newton was divided by the surface area (l × b) at
the fracture of the specimen, to obtain values in Graph 1: Comparison of the mean bond strengths of all the
megapascal (MPa = Newton/mm2). groups

Table 1: Comparison of the mean and standard deviation values of microtensile bond strength (megapascal) among the groups
Mean±SD
Group I: Group II: Blood + Group III: Blood + ViscoStat Group IV: Blood + ViscoStat
Control (n=18) ViscoStat Clear (n=18) Clear + 6.5% GSE (n=18) Clear + 10% Hesperidin (n=18)
Microtensile BS (MPa) 31.84±0.27 15.62±0.98 30.45±0.92 31.23±0.79
GSE: Grape seed extract, BS: Bond strength

468 Journal of Conservative Dentistry and Endodontics | Volume 26 | Issue 4 | July-August 2023
 Manihani, et al.: Effect of collagen cross‑linkers on microtensile bond strength after contamination
The majority of the samples showed adhesive failures. In
the SEM analysis, the control group [Figure 2a] depicted
the maximum number of dentinal tubules obliterated with
resin plugs. In Group II [Figure 2b], some dentinal tubules
were open, and a few were obliterated. In Groups III and
IV [Figure 2c and d], the majority of the tubules were
obliterated with resin plugs.
DISCUSSION
Adequate adhesion necessitates a clean substrate.[11] Various
isolation methods are employed to prevent moisture
contamination. However, avoiding contamination is a
challenge. Such is the case in many clinical scenarios where
the cavity margins extend below the level of the gingiva.[2]
Blood and gingival crevicular fluid are notorious in such cases.
This contamination can either be caused due to inflammation
or operator‑related factors. The success of the restoration in
such cavities relies majorly on the adaptation of the restorative
material. From a clinical point of view, microleakage, leading
to secondary caries in a subgingival cavity, is a failure.
HAs are used with or without a retraction cord to control
bleeding. They work by either of the three mechanisms:
vasoconstriction, protein coagulation, or styptics.[12]
Vasoconstrictors work by narrowing the blood vessels.
Hence, tissue perfusion is controlled. Aluminum chloride is
one of the most widely employed HAs nowadays. It controls
bleeding by constricting blood vessels. It also removes
tissue fluids. Hence, ViscoStat Clear (25% aluminum
chloride) was used in this study.
Several studies indicate lowered BSs of self‑etch bonding
agents due to HA contamination. Total‑etch adhesives,
a b
c d
Figure 2: Scanning electron micrographs of debonded
specimens. (a) Group I (Uncontaminated group),
(b) Group II (Contaminated with blood and hemostatic
agent), (c) Group III (Grape seed extract), and (d) Group IV
(Hesperidin)
Journal of Conservative Dentistry and Endodontics | Volume 26 | Issue 4 | July-August 2023
on the other hand, are also affected (Caesar Rogerio).[13]
However, they fare better than SEAs postcontamination.[14] A
universal self‑etch bonding agent Single Bond Universal (3M
ESPE St. Paul, MN, USA) was used in this study.
Research suggests that MMPs are to be blamed for collagen
degradation. They are endogenous proteins residing in
dentin in an inactive state. MMPs 2, 3, 8, 9, and 11 are found
in dentin.[15] The two ions that they primarily depend on are
zinc and calcium. They are activated by a low pH, which
can be either caused due to demineralization due to caries,
application of HAs, or application of dental adhesives.[16,17]
These MMPs with cathepsins destroy collagen through
hydrolysis. The fundamental concept of hybrid layer
bonding is through the anchorage of resin by collagen. The
disintegration of the collagen leads to the failure of the
bond.
Wet bonding advocates that the tooth surface must be left
moist before bonding to prevent the collapse of collagen
fibrils. The destruction of collagen fibrils is the causative
factor for adhesive failures.
Many initiatives have been taken for compensation of
lowered BS postcontamination. Some authors advocate
conventional methods such as rinsing, drying, and
repriming when contamination occurs after the priming.[18]
Nevertheless, there may be a loss of BS.
The literature documents the use of CCLs to overcome this
decrease.[19] They are naturally extractable bioflavonoids
that yield a cyanidin on boiling with acid. Research has
indicated that they enhance the cross‑linking of collagen.
They are among the many secondary metabolites obtained
in the plant kingdom. They own a great structural diversity
with variable monomer molecules. They are differentiated
by those containing either catechin or ent‑catechin.
Moreover, they may also have either epicatechin or
ent‑epicatechin. Their interflavanoid bonds and degree of
polymerization might be different.[8] They are advantageous
in binding to proline‑rich proteins. The quality of collagen,
which is the major organic constituent of the tooth, can be
very well enhanced by these bioflavonoids. Studies show
that naturally occurring CCLs depict a positive influence
on collagen cross‑linking.[6‑10,20,21] Examples include sodium
ascorbate, GSE, hesperidin, and riboflavin 5’‑phosphate.
Other cross‑linkers such as tannic acid, cocoa seed extract,
glutaraldehyde, and chlorhexidine have also been used.
In this study, blood and HA were the contaminants. They
were applied for 1 min each and were rinsed for 1 min.
Some authors (Brauchli et al., in 2010) advocated that
rinsing for 1 min was sufficient to effectively remove
the contaminants.[18] The results of this study depicted
a decrease in BS postcontamination by blood and HA.
469
Manihani, et al.: Effect of collagen cross‑linkers on microtensile bond strength after contamination
The mean BS value of the noncontaminated group was
31.84 ± 0.27 MPa. This was reduced after blood, and HA
was applied to obtain a mean BS of 15.62 ± 0.98 MPa. This
decrease can be attributed to two factors, i.e. the low pH
of the HA which activates the MMPs and the removal of
the smear layer which hinders the hybridization process
required for bonding using SEA.
Moreover, the HA replaces calcium in hydroxyapatite. The
result is an insoluble compound. Hence, demineralization
becomes incomplete. Therefore, the BS of SEAs is reduced.[22]
In Group III, GSE was employed after blood and ViscoStat
Clear contamination. The mean BS of Group III was
30.45 ± 0.92 MPa. This was greater than the contaminated
group but less than the control group. Hence, it can be
said that GSE can be used to overcome the loss of BS
postcontamination. PA found in GSE is condensed tannin. Its
main mechanism of action can be explained by a hydrogen
bond formation between proteins. This occurs among
amide carbonyl and phenolic hydroxyl (Han et al., in 2003).[7]
Moreover, it also inhibits collagenase. In addition, it also
increases the conversion of soluble to insoluble collagen.
Another mode of its action can be either due to covalent or
ionic or hydrophobic mechanisms. In a systematic review
and meta‑analysis, it was stated that PA/GSE was the most
efficient natural cross‑linker to date, in preserving the BS
even after aging.[23]
Hesperidin (hesperetin‑7‑O‑rutinoside) was used in
Group IV. It is a glycoside flavonoid and a naturally
occurring CCL. A study by Hiraishi et al., in 2011, showed
it to preserve dentin collagen.[21] Another study by Islam
et al., in 2012, concluded that hesperidin inhibited collagen
degradation and led to the arrest of demineralization in
radicular dentin.[16] The mean µTBS of this group was
31.23 ± 0.79 MPa. This BS was marginally more than in
Group III, i.e. GSE.
The main point of distinction between the two CCLs is their
molecular weight. Hesperidin has a low molecular weight.
On the contrary, GSE has a high molecular weight.[24]
Both the CCLs are amphiphilic.[25] They are both hydrophobic
and hydrophilic. The hydrophilic part of GSE is capable
of inducing links with collagen. In contrast, hesperidin’s
hydrophobic property is more marked and has a stronger
association with collagen fibrils.
The specimens were subjected to µTBS testing. Its
advantages include even stress distribution and evaluation
of small samples. The majority of the samples showed
adhesive failures.
The limitation of the study is that the results need to be
validated in a simulated oral environment.
470 Journal of Conservative Dentistry and Endodontics | Volume 26 | Issue 4 | July-August 2023
CONCLUSIONS
1. Contamination with blood and ViscoStat Clear
decreased the µTBS of composite resin and dentin with
Single Bond Universal Adhesive
2. The µTBS of contaminated dentin was significantly
increased when the CCLs‑PA (6.5% GSE) and 10%
hesperidin were applied with Single Bond Universal
Adhesive
3. Hesperidin 10% showed marginally higher µTBS than
PA (6.5% GSE) with Single Bond Universal Adhesive.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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