UNIVERSIDADE ESTADUAL PAULISTA

“JÚLIO DE MESQUITA FILHO”

Instituto de Ciência e Tecnologia

Campus de São José dos Campos

ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2023.e3869

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Inﬂuence of two natural cross-linkers on microtensile bond strength

durability – An in vitro study

Inﬂuência de dois reticuladores naturais na durabilidade da resistência de união à microtração – Um estudo in vitro

Lavanya ANUMULA

1,2

, Sindhu RAMESH

, Venkata Suneel Kumar KOLAPARTHI

, Rama Krishna ALLA

, Kiranmayi GOVULA



1 - Narayana Dental College and Hospital, Department of Conservative Dentistry and Endodontics. Nellore, A.P. India.

2 - Saveetha Dental College and Hospital, Conservative Dentistry and Endodontics. Chennai, Tamil Nadu, India.

3 - Saveetha University, Saveetha Institute of Medical and Technical Sciences, Saveetha Dental College and Hospital, Department of

Conservative Dentistry and Endodontics. Chennai, Tamil Nadu, India.

4 - Narayana Dental College and Hospital, Department of Oral Medicine and Radiology. Nellore, A.P. India.

5 - Vishnu Dental College, Department of Dental Materials. Bhimavaram, Andhra Pradesh, India.

6 - Narayana Dental College and Hospital, Department of Conservative Dentistry and Endodontics. Nellore, A.P. India.

How to cite: Anumula L, Ramesh S, Kolaparthi VSK, Alla RK, Govula K. Inuence of two natural cross-linkers on microtensile bond

Strength durability – An in vitro study. Braz Dent Sci. 2023;26(4):e3869. https://doi.org/10.4322/bds.2023.e3869

ABSTRACT

Objective: to investigate the effect of two natural cross-linkers on microtensile bond strength (µTBS) and evaluate their inuence on the

durability of the resin dentin bonds. Material and Methods: the

Moringa oleifera

and

Centella asiatica

plant extracts were qualitatively

tested with high-performance thin layer chromatography (HPTLC) for the presence of phenols. The phenolic content ranged from

27 to 30 gallic acid equivalents (GAE), µg/mg of dry weight. After etching, two concentrations (5% and 1%) of these two extracts were

prepared and used as pretreatment liners on dentin. They were applied for a min. After restoration with resin composite, dentin resin beams

were prepared. The study groups were 5%

Moringa

, 1%

Moringa

Centella

, and control (without cross-linker application).

For each group, half of the samples underwent µTBS testing after 24 hours, while the remaining half were immersed in articial

saliva to assess the bond’s longevity after 6 months of ageing. Statistical analysis was performed using one-way ANOVA followed by

Tukey’s post hoc test. Results: both 5% and 1%

Moringa

showed a signicant difference (p<0.05) compared to the other groups at

both intervals. However, after ageing, the specimens in the control and 1%

Centella

groups resulted in a signicant decrease in µTBS.

Conclusion: overall, both concentrations of

Moringa

(5% and 1%) were effective in stabilising the bond during both intervals.

KEYWORDS

Collagenase; Endopeptidases; Matrix metalloproteinase; Moringa; Phenols.

RESUMO

Objetivo: investigar o efeito de dois reticuladores naturais na resistência de união (µTBS) à microtração e avaliar sua inuência

na durabilidade da adesão da resina à dentina. Material e Métodos: extratos das plantas Moringa oleifera e Centella asiatica

foram qualitativamente testados através de cromatograa em camada na de alta performance (HPTLC) para a presença

de fenóis. O conteúdo fenólico alcançou entre 27 a 30 equivalentes de ácido gálico (GAE), µg/mg de peso seco. Após o

condicionamento, duas concentrações (5% e 1%) dos extratos foram preparadas e utilizadas como forros de pré-tratamento

em dentina. Eles foram aplicados por um minuto. Após a restauração com resina composta, palitos de dentina e resina foram

preparados. Os grupos foram 5% Moringa, 1% Moringa, 5% Centella, 1% Centella e controle (sem aplicação de reticulador).

Para cada grupo, metade das amostras foram submetidas ao teste µTBS após 24 horas, enquanto a outra metade foi imersa em

saliva articial para avaliar a longevidade adesiva após 6 meses de envelhecimento. Foi realizada análise estatística através de

ANOVA 1-fator, seguido do teste post hoc de Tukey. Resultados: ambas as concentrações de 5% e 1% de Moringa demonstraram

diferença signicativa (p<0.05) comparadas aos outros grupos em ambos os intervalos. No entanto, após o envelhecimento,

os espécimes dos geupos controle e 1% de Centella resultaram em uma redução signicativa de µTBS. Conclusão: no geral,

ambas as concentrações de Moringa (5% e 1%) foram efetivas em estabelecer a adesão em ambos os intervalos.

PALAVRAS-CHAVE

Colagenase; Endopeptidases; Matriz de metaloproteinase; Moringa; Fenóis.

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Anumula L et al.

Influence of two natural cr oss-linkers on microtensile bond str ength durability – An in vitro study

Anumula L et al.

Inﬂuence of two natural cross-linkers on microtensile bond

strength durability – An in vitro study

INTRODUCTION

The eld of esthetic dentistry has undergone a

remarkable transformation with the advancements

in adhesive materials, particularly the resin based

composite restoration. These innovations have

not only made these restorations more user

friendly but have also contributed to soaring

popularity in recent years [1,2]. The continuous

development of adhesive materials has brought

about a paradigm shift, thus facilitating reliable

bonding of resin composite to both enamel and

dentin, resulting in esthetically pleasing and

durable restorations [3]. Moreover, the advent

of minimally invasive operating methods has

further enhanced the clinical performance and

longevity of these restorations, ensuring optimal

oral health outcomes [4].

Contemporary adhesive technology has

made signicant strides in achieving immediate

bonding, regardless of the approach used. The

durability of the adhesive bond in restorative

dentistry is crucial for the restoration’s longevity

and clinical performance. However, challenges

persist in maintaining the durability and the bond

strength of these adhesives gradually decreases

[5,6]. The causes identied for the degradation

of the bond between resin and dentin are water

sorption, hydrolysis of the hydrophilic resin

component and activation of endogenous dentin

matrix metalloproteinases (MMPs) [7].

MMPs are zinc- and calcium-dependent,

host-derived collagenolytic endopeptidases that

are primarily activated through proteolytic cleavage

of their proenzyme (Zymogen) forms, converting

them into active enzymes. The activation of

MMPs can occur by enzymatic activation or by an

acidic pH. The acidic environment occurs during

pathological conditions such as caries or during

restorative procedures [8]. The acidic pH can

cause conrmational changes in the proenzyme

structure, leading to the exposure of the active

site and subsequent activation of MMPs [9].

The hybrid layer, a critical transition zone

between the resin in the bonding agent and

collagen in dentin, distributes the stresses

generated during functioning and prevents the

bond from breaking down over time [10]. The

stability and integrity of collagen brils form the

structural foundation of the hybrid layer and are

essential for the durability of the resin-dentin

bond. The collagen fibers with incomplete

inltration of resin below the hybrid layer are

referred to as exposed collagen, which has several

implications for the stability and longevity of

the bond [11]. These exposed collagen bers

may put the hybrid layer at risk due to exposure

to MMPs, which trigger the collagenolytic

activity and weaken the bond strength [12].

Armstrong provided the first transmission

electron micrographs of the deteriorating hybrid

layer, demonstrating the loss of insoluble collagen

fibrils [13]. The inhibition of these proteases

or improving the collagen structure resistance

to these enzymes is the primary approach for

preventing the degradation of collagen bers in

hybrid layers [14].

To address this concern, the use of cross

linking agents has gained attention as a potential

strategy to enhance bond strength and improve

durability of adhesive surfaces. The inherent

collagen cross-linking contributes to the stability,

strength, and function of the dentin matrix [15].

The application of exogenous collagen cross-linkers

(Dentin biomodification) induces additional

inter and intramolecular cross-links of collagen

that aid not only in strengthening the dentin

matrix but also decreasing the biodegradation

rate [16,17]. Several synthetic and natural

collagen cross-linkers have been extensively

studied to improve the mechanical properties

of demineralized dentin [18-20]. Thus, the

application of external cross-linkers is an attempt

to stabilize the exposed collagen below the hybrid

layer to benet the durability of the bond.

Glutaraldehyde is a well-known synthetic

cross-linking agent for intermolecular covalent

cross-linking, although concerns regarding

cytotoxicity have restricted its use [21]. Natural

plant extracts rich in potential polyphenol

molecules promote cross-linking between the

fibrils [15] and stabilize collagen [22]. They

form effective hydrogen bonds between collagen

polypeptides, making them less susceptible to

collagenase attack and inhibiting MMPs with their

antioxidant property [23]. Natural cross-linkers

have always been a better choice over synthetic

agents as they also demonstrate MMP inhibition

with a high level of biocompatibility [22].

Adhesion to tooth structure should be

simple to achieve, provide marginal seal, have

clinical durability, and retentive strength.

Microtensile bond strength test (immediate and

aging) is the most widely accepted method to

measure retention of dental resin composite [24].

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Anumula L et al.

Influence of two natural cr oss-linkers on microtensile bond str ength durability – An in vitro study

Anumula L et al.

Inﬂuence of two natural cross-linkers on microtensile bond

strength durability – An in vitro study

The reduced cross-sectional area of specimens

in µTBS allows faster water diffusion through

the hybrid layer, thus mimicking the in vivo

conditions [25].

The plant extracts of

Moringa

and

Centella

are rich in phenols and avonoids. In our earlier

study, the effect of 5%

Moringa

, 1%

Moringa,

Centella and

Centella

in reducing the collagen

degradation and loss of dry mass was observed.

In the pretreated groups, there was a decrease in

collagen degradation, as evidenced by the reduced

release of ICTP and less loss of dry mass. This stands

in contrast to the untreated group. The efciency of

these cross-linkers was dose-dependent i.e, higher

concentrations proved better at inhibiting MMPs,

thus reducing collagen degradation. (unpublished).

The present manuscript serves as a continuation

of that research building upon the preliminary

ndings and expanding the investigation to assess

the effect of

Moringa

and

Centella

on the bond

strength to dentin to characterize its clinical

performance. While the detailed results and

analysis of the previous study are not included

in this manuscript, we have incorporated the

relevant ndings into our current research design

and interpretation. Therefore, this study aimed to

evaluate the µTBS of the resin composite to the

dentin, pretreated with the same concentrations

Moringa

and

Centella

(5% and 1%). The study

aimed to test three hypotheses each focusing

on different aspects of the influence of natural

cross linkers on the bond strength of dentin

resin interface. The first hypothesis proposed

was (a) the natural cross-linkers,

Moringa

and

Centella

are rich in polyphenol content. The

second hypothesis (b) that the pretreatment of

demineralized dentin with

Moringa

and

Centella

before application of dentin-bonding agent would

preserve the bond strength of the dentin–resin

interface even after aging. The third hypothesis

in bond strength with different concentrations

of cross linkers. Hypothesis “a” and “b” were

alternate hypotheses, while hypothesis “c” served

as the null hypothesis.

MATERIAL AND METHODS

Plant collection and preparation of plant extract

The fresh leaves of

Moringa oleifera

and

Centella asiatica

were destalked, shade

dried, and powdered using an electric blender.

The shade-dried powder was subjected to

hydroalcoholic extraction by cold percolation

method. About 10g of the shade-dried and

coarsely powdered plant material was soaked in

100 ml of 70% ethanol solution for 24h. After

24h, the extract was ltered using Whatman No.1

lter paper (Cytiva, United states). The lter cake

was collected and dried at 37°C. The dried extract

was stored at 4°C and used for HPTLC analysis.

The extracts were prepared to the required

concentration using dimethyl sulfoxide (DMSO).

High-performance thin layer chromatography [

]

High-performance thin layer liquid

chromatography (HPTLC) was performed on a

10x10 cm preactivated silica gel 60F 254 plate. The

extracts of the

Moringa oleifera

and

Centella asiatica

and standard gallic acid of the required concentration

were prepared and spotted using CAMAG Linomat

5 applicator (Switzerland). They were allowed to

develop in a twin trough chamber of size 10x10 cm

at 25±5°C which was kept saturated with the solvent

system Chloroform: Ethyl Acetate: Formic Acid at a

ratio of 2.5:2.0.:0.8 in an ascending mode manner

under room temperature. The solvent system was

found suitable for gallic acid, which was selected

by trial and error and conrmed through its specic

Rf values. After development, a pencil was used to

mark the solvent front, and a hair dryer was used

to dry the plates. The densitometric scanning of

the developed plates was done at 280nm using

scanner 3 and photo-documented using CAMAG

reprostar 3 (Switzerland) at 254nm and 366nm. The

quantication of gallic acid in

Moringa oleifera

and

Centella asiatica

was conrmed, and the results were

expressed as mg of gallic acid per milligram of extract.

Specimen preparation

Fifteen sound non-carious human third

molar teeth, extracted from people aged 18 to

45 years, were collected from the Department of

Oral Surgery Narayana Dental College, following

approval from the Institutional Ethical Committee

(IEC/NDCH/2021/SEP/P-24). The teeth were

cleaned, and stored in distilled water containing

0.5% Chloramine T solution. The teeth were used

within a month of extraction.

The teeth were embedded in rectangular

molds of self-cure acrylic resin. The occlusal

enamel was removed and a at dentin surface was

exposed under water cooling using a slow diamond

cutter (model: Struers- Minitom, United States).

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Anumula L et al.

Influence of two natural cr oss-linkers on microtensile bond str ength durability – An in vitro study

Anumula L et al.

Inﬂuence of two natural cross-linkers on microtensile bond

strength durability – An in vitro study

An 8-fluted carbide bur produced a uniform

smear layer on the exposed dentin surfaces. The

teeth were randomly allocated to 4 test groups

(n=3) and one control group. The 4 test groups

were 5%

Moringa oleifera

, 5%

Centella asiatica

Moringa oleifera

and 1%

Centella asiatica

. All

the surfaces were etched with 37% phosphoric

acid for 15 seconds and rinsed thoroughly. To

prepare the dentin surfaces, the test solutions

were applied as a pretreatment liner using an

applicator brush for one minute. Any excess

solution was blotted and the surface was left to

dry. The universal bonding agent (Ivoclar Tetric

N-Bond Universal) was applied and rubbed for

15 seconds with a micro brush. After a dwelling

period of 2min, it was air dried with a gentle

air blow. There was no pretreatment liner

application in the sample teeth that belonged to

control group, and the bonding procedure was

followed after etching. After applying the bonding

agent, all the samples were cured for 20 seconds

(Ivoclar Bluephase N polywave LED curing light).

Around 4 mm of resin composite (Ivoclar Tetric

N Ceram) was incrementally built and cured on

entire dentin surfaces.

The samples were stored in distilled water

for 24 hours and later subjected to longitudinal

sectioning with a slow diamond (model:

Struers-Minitom, United States) under copious

irrigation. Longitudinal beams of dentin- resin

composite of around 1 x 1mm were prepared.

Around 8 to 12 beams were obtained per tooth.

The beams from one group were randomly

divided into two sub test groups. Each sub group

had ten (n=10) samples. One sub group was

tested immediately for bond strength, while the

other sub group was stored in articial saliva

for six months to evaluate the impact of aging

on bond strength. Thus, a total of 50 samples

were tested immediately and another 50 after

six months. The storage media (articial saliva)

was replaced once in 15 days.

Microtensile bond strength (µTBS) testing

The bond strength testing was performed

after one day and six months of storage. The

exact dimension of each beam was measured

with a digital caliper to determine the bond

area accurately. The specimens were xed, one

at a time, to a customized jig (Geraldeli’s Jig)

using cyanoacrylate glue, ensuring that the

micro-stick was parallel to the direction of the

test block and mounted on a Universal Testing

Machine (Instron E300). A tensile load was

applied until failure at a crosshead speed of

1 mm/min. The µTBS in MPa at fracture was

calculated by dividing the load to fracture by

the surface area of the cross-section of the

micro-sticks (bond area in mm

) according to the

formula MPa = N/ (a x b) where a and b are the

dimensions of the beam at resin dentin interface.

(Width and thickness).

Fracture mode analysis

The fractured beams were observed under

a stereomicroscope (Lawrence Mayo Model.

NO: LM-52-3621) at 80X magnification with

transmitted LED light to determine whether the

mode of failure was an adhesive failure (A),

Mixed (M), cohesive failure in dentin (CD) or a

cohesive failure in resin composite (CC).

SEM analysis

The fractured beams were cleaned with

ethanol to remove any loose debris. The sample

was later mounted on the brass stubs using

carbon tape. Then the samples were placed

in a vacuum chamber for sputtering and were

platinum coated for 40 seconds. The images were

captured in a set of magnications to study the

fracture site (FE-SEM IT800, JEOL).

Statistical analysis

The obtained data were statistically analyzed

using unpaired t-test to examine if there was a

difference in µTBS between the samples before

and after the storage period (after 24 hours

and 6 months). One-way ANOVA test was used

to determine the effects of the cross linker and

storage time on µTBS among different groups.

The multiple pairwise comparisons among

group means were made using Post hoc Tukey’s

test. Statistical signicance was set at p = 0.05.

All data analysis was performed by SPSS 20.0

software package (SPSS, Chicago, IL, USA).

RESULTS

Assessment of phenols

Based on the results obtained from HPTLC

analysis, it was found that the Rf value of

standard gallic acid and the Rf value of a peak

in the plant extract was identical, conrming

the presence of gallic acid in the plant extract.

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Anumula L et al.

Influence of two natural cr oss-linkers on microtensile bond str ength durability – An in vitro study

Anumula L et al.

Inﬂuence of two natural cross-linkers on microtensile bond

strength durability – An in vitro study

The standard curve of gallic acid was considered

and the quantication was estimated as gallic

acid equivalents (GAE), µg/mg of dry weight.

All determinations were performed in triplicate

(n = 3). The amount of gallic acid in the

Moringa

was 29.93 µg of GA/mg of extract, and in

Centella

extract, it was found to be 27.97 micrograms per

milligram of plant extract. Figure 1 and Figure 2

represent the photo document 254nm and 366nm

and the Peak area of hydroalcoholic extract of

Moringa

(10µl) and

Centella

(10µl), respectively.

µTBS

The mean µTBS of all groups at different time

intervals are given in Table I. µTBS values at 1 day

and 6 months for each group were compared using

unpaired t-test, and it did not show any signicant

difference except for Group 4 (1%

Centella

Figure 1 - Chromatograms obtained from separation of plant extracts and peak areas of hydroalcoholic extract of

Moringa Oleifera

in HPTLC analysis.

Figure 2 - Chromatogram and peak areas of hydroalcoholic extract of

Centella asiatica

in HPTLC analysis.

Table I - Comparison of mean µTBS values of all tested groups at two-time intervals

GROUPS 1 DAY 6 MONTHS UNPAIRED T-TEST Bond strength % change (related to time)

5% MORINGA 23.45±5.89 28.60±7.88 0.402(NS) 22.3% ˄

5% CENTELLA 14.28±4.04 16.23±4.80 0.340(NS) 14.3% ˄

1% MORINGA 18.62±5.31 18.79±5.40 0.944(NS) 0.912% ˄

1% CENTELLA 14.81±3.75 10.09±4.80 0.025* ˗ 31.8% ˅

CONTROL 14.43±5.26 10.57±4.10 0.084(NS) ˗ 26.4% ˅

ONE WAY ANOVA <0.001* <0.001*

˄ indicates increase in bond strength %. ˅ decrease in bond strength %. p<0.05* signiﬁcant. (NS) = non-signiﬁcant.

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Anumula L et al.

Influence of two natural cr oss-linkers on microtensile bond str ength durability – An in vitro study

Anumula L et al.

Inﬂuence of two natural cross-linkers on microtensile bond

strength durability – An in vitro study

Figure 3 shows the difference in mean µTBS

values of all groups at day 1 and 6 months.

One-way ANOVA analysis of mean µTBS values

in all the groups showed a signicant difference

(p=0.001) at 1-day and 6-month intervals.

Group 1 (5%

Moringa

) showed the highest mean

values of bond strength at both intervals. Group 4

(1%

Centella

) and 5 (Control) showed the least

values. 5%

Moringa

showed an appreciable increase

in bond strength at the 6-month interval (22.3%),

whereas 1%

Centella

showed a decrease in bond

strength (31.8%) similar to control (26.4%).

After conducting an ANOVA, a significant

difference was found in the group comparison.

To further investigate this, the Post hoc Tukey

test was utilized (refer to Table II). At the 1-day

interval, 5%

Moringa

showed a significant

difference compared to

Centella

5%, 1%, and

control group. At 6 months interval, there

was a considerable difference between 5%

Moringa

with all the other groups.

Moringa

too showed a significant difference when

compared to

Centella and control group

(at 6 month interval).

Stereo-microscopic analysis:

The failure mode of fractured beams was

classified as Mixed, Adhesive or Cohesive

(in Dentin or Resin Composite). Table III

represents the observations made and analysis

with Fisher’s exact test. There was no signicant

difference in failure modes among all groups

when compared at 1-day and 6-month intervals.

Moringa

showed 70% adhesive failure when

compared to control with 100% adhesive failure

at 6-month intervals. But this nding was not

statistically signicant.

Figure 3 - Mean µTBS values of all groups at 1-day and 6 months

intervals.

Table II - Post hoc Tukey – to test signiﬁcance in between groups (p<0.05* signiﬁcant)

INTERVALS GROUPS pVALUE

1 day

5% MORINGA

5% CENTELLA 0.001*

1% MORINGA 0.201(NS)

1% CENTELLA 0.003*

CONTROL 0.002*

5%CENTELLA

1% MORINGA 0.297(NS)

1% CENTELLA 0.999(NS)

CONTROL 1.000(NS)

1% MORINGA

1% CENTELLA 0.428(NS)

CONTROL 0.331(NS)

1%CENTELLA CONTROL 1.000(NS)

After 6 months

5% MORINGA

5% CENTELLA <0.001*

1% MORINGA 0.002*

1% CENTELLA <0.001*

CONTROL <0.001*

5% CENTELLA

1% MORINGA 0.84(NS)

1% CENTELLA 0.116(NS)

CONTROL 0.172(NS)

1% MORINGA

1% CENTELLA 0.009*

CONTROL 0.015*

1%CENTELLA CONTROL 1.000(NS)

a,c,d

representing the statistical signiﬁcance between the groups at both intervals. Where as

b,e,f

represents the signiﬁcance at 6 months

interval only. (NS) = no signiﬁcance.

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Anumula L et al.

Influence of two natural cr oss-linkers on microtensile bond str ength durability – An in vitro study

Anumula L et al.

Inﬂuence of two natural cross-linkers on microtensile bond

strength durability – An in vitro study

SEM analysis

The SEM image of the fracture site revealed

distinct features indicative of the nature of the

bond failure. Figure 4A-D represents the SEM

images of beams that had adhesive failure and

cohesive failures. In a cohesive failure, both the

resin composite and dentin fracture surfaces show

similar characteristics, indicating that the failure

occurred within the material itself (Figure 4C).

In contrast, adhesive failure results in a distinct

separation between the resin composite and

dentin, indicating a failure at the interface. The

tubules within the dentin are visible and appear

to be lled or plugged (Figure 4A), suggesting an

attempt to bond the resin composite and dentin.

Table III - Fisher’s exact test analysis of the mode of failure of dental beams in all test groups at both intervals. (within groups and in between groups)

GROUPS

1 DAY 6 MONTHS

P vAlUe

ADHeSIve

FAIlURe

COHeSIve FAIlURe IN

DeNTIN /ReSIN

ADHeSIve

FAIlURe

COHeSIve FAIlURe IN

DeNTIN /ReSIN

5% MORINGA 9(90) 1(10) 7(70) 3(30) 0.264(NS)

5% CENTELLA 9(90) 1(10) 8(80) 2(20) 0.531(NS)

1% MORINGA 9(90) 1(10) 9(90) 1(10) 1.000(NS)

1% CENTELLA 9(90) 1(10) 8(80) 2(20) 0.531(NS)

CONTROL 8(80) 2(20) 10(100) 0(0) 0.136(NS)

FISHER’S EXACT TEST (p value) 0.944(NS) 0.424(NS)

(NS) = no signiﬁcance.

Figure 4 - SEM images: (A) representing adhesive failure- tubules with plugged dentin; (B) adhesive failure - tubules surrounded by exposed collagen;

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Anumula L et al.

Influence of two natural cr oss-linkers on microtensile bond str ength durability – An in vitro study

Anumula L et al.

Inﬂuence of two natural cross-linkers on microtensile bond

strength durability – An in vitro study

DISCUSSION

The results of the present study partially

supported hypothesis regarding the natural

cross-linkers,

Moringa

and

Centella

, being

rich in polyphenols (hypothesis ‘a’). However,

the effectiveness of preserving bond strength

through the pretreatment of demineralized

dentin with these extracts varied depending on

the concentration used, leading to the rejection

of hypothesis ‘b’ and ‘c’.

Upon testing after a day, the mean bond

strength values were similar for most groups except

for 5%

Moringa

, which showed an appreciable

increase in bond strength. Additionally, 1%

Moringa

showed slightly higher values than the

control group. Notably

Moringa

demonstrated

the ability to stabilise the bond strength at low

concentrations while increasing it at higher

concentrations. This suggests that

Moringa,

a potent cross linker, preserves collagen fibre

integrity and inhibits MMPs. On the other hand

Centella

, maintained stable bond strength

after ageing in contrast to the lower concentration

of 1%. However, no signicant increase in bond

strength was observed in 5%

Centella

and 1%

Moringa

(as depicted in Figure 3). This indicates

that while

Centella

may possess cross linking

properties, it may not function as an effective MMP

inhibitor like

Moringa

. It can be hypothesized

that lack of cross linking agent in the control

group, may have increased the likelihood of bond

degradation and weakening over time.

Our previous study examining telopeptide

ICTP release showed similar ndings. Among the

tested agents, 5%

Moringa

exhibited signicant

efciency in inhibiting MMPs and cross linking

the collagen, as evidenced by reduced telopeptide

release from the dentin beams. Therefore,

future research could focus on investigating the

molecular nature of these two natural extracts.

Although both extracts contained substantial

phenolic content, the difference in their ability

to preserve bond strength could be attributed to

variations in molecular structure, complexity of

the molecule, and penetration. A similar nding

was reported by Aguiar et al. [16], where each

plant contained diverse polyphenol with different

monomeric constituents, linkages, relative and

absolute concentrations etc., and their effect

on the dentin matrix depended on the chemical

composition and concentrations of these complex

phytoconstituents.

Preserving the structural integrity of collagen,

within the hybrid layer unimpregnated by resin,

is crucial for the durability of resin dentin bonds.

Polyphenols, including avonoids, play a vital

role in preserving the demineralised collagen

matrix and increase the resin-dentin bond

strength over time. Flavonoids, (a subgroup of

polyphenols) found in plant derived cross linkers

possess amphiphilic properties and interact

physically with collagen, promoting cross linking

and stabilising the collagen network, thereby

reducing biodegradation [27]. Chung et al. [28]

demonstrated that for the collagen to be cleaved,

it has to be primarily unwound to allow the

enzymes’ active site to cleave individual chains.

Thus, the role of cross linkers is very vital. The

formation of exogenous collagen cross links

improves the resistance of the collagen matrix to

enzymes . Therefore, biomodication of dentin

with collagen cross linkers contributes to a secured

matrix network and a stable hybrid layer in dentin

bonding. Dentin when treated with natural

agents (plant-derived) such as proanthocyanidin,

hesperidin, and EGCG have shown increased

collagen resistance to degradation even when

challenged with bacterial collagenases [29,30].

Polyphenols also chelate zinc, which is

vital for maintaining functional, active sites

of metalloproteinases and inhibiting MMP

activity [31]. Consequently, high level

of biocompatibility and effective collagen

cross-linking capacity of polyphenols make them

favourable MMP inhibitors. Also, Polyphenols,

occurring naturally in plants, have been

extensively studied for their capacity to cause

MMP inhibition [32]. Plant avonoids in whole

plant extracts have also been investigated

and employed topically for inammatory skin

disorders for MMP inhibition [33].

Thus, an attempt was made to preserve

the integrity of the exposed collagen within the

hybrid layer by evaluating the efcacy of 2 natural

extracts,

Moringa Oliefera

and

Centella Asiatica

rich in polyphenols, as collagen cross-linkers

and MMP inhibitors on dentin’s microtensile

bond strength with resin composite. The results

indicated that both the natural extracts at higher

concentrations, acted as efcient cross-linkers

stabilising the resin dentin bond (The bond strength

values remained unchanged even after aging).

Moringa,

in particular, demonstrated superior

performance stabilising the bond even after aging

and exhibiting increased bond strength after

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Anumula L et al.

Influence of two natural cr oss-linkers on microtensile bond str ength durability – An in vitro study

Anumula L et al.

Inﬂuence of two natural cross-linkers on microtensile bond

strength durability – An in vitro study

6 months (Figure 3) with a 5% concentration,

which could be attributed to its cross linking

property. This study utilised a universal bonding

agent in the etch and rinse mode, following a

systematic review [34], suggesting this technique

improves microtensile bond strength.

Microtensile bond strength test is currently

recommended for assessing the retention of resin

composite restoration, especially after subjecting

the specimens to a durability challenge [24].

It is calculated as the tensile load at failure divided

by the cross-sectional area of the bonded surface.

Though the preparation of micro specimens is

a technique-sensitive, µTBS offers versatility,

allowing multiple samples to be obtained from a

single tooth. This test leads to more inventive study

setups, better-controlled substrate variables, and

economical use of teeth [24]. The test provides

valuable information about biodegradation

mechanism in in-vitro studies, even though

these conditions may not occur separately in

an intraoral condition. In this study, the most

appropriate statistical approach was to conduct an

unpaired T-test due to the nature of micro tensile

testing, where the initial sample was destroyed

and not reused after ageing.

It is worth noting that the type of MMP

inhibitor, its concentration, and aging period

can inuence the impact of MMP inhibitors on

bond strength [35]. Hence, this study examined

the impact of MMP inhibitors on µTBS for up

to 6 months utilising different concentrations

cross linkers. Storage in water to simulate

aging is one of the most common artificial

technique to predict the behaviour of resin-based

restorative materials [36] because the presence

of water is critical for their deterioration of

material [37]. To expedite the aging process,

the specimens are sectioned into sticks, reducing

the time required for water diffusion through the

exposed resin dentin interface [38,39] during

durability testing. In this study, articial saliva

was used as aging media. Another method

used to study aging and bond durability is

thermocycling [40], a thermal fatigue test which

simulates thermal changes in the oral cavity

caused by eating, drinking, and breathing [41].

The alteration in the temperature induces

tedious contraction/expansion stresses at the

tooth-material interface [5], resulting in crack

propagation along the bonded interface.

The µTBS of 5%

Moringa

reported in

this study was comparable with the results of

Saffarpour et al. [42], who reported almost similar

µTBS with chlorohexidine. Zheng et al. [43]

documented slightly more µTBS with dentin

treated with chemical MMP inhibitors, such as

chlorohexidine and Galardin. Nagpal et al. [44]

compared the inuence of natural and synthetic

matrix metalloproteinase inhibitors on dentin

collagen preservation and their effect on long-term

bond strength using total-etch adhesive. They

reported that the natural MMP inhibitors increase the

bond strength by improving resistance to collagen

degradation. Al-Ammar et al. [18] concluded that

glutaraldehyde and grape seed extract promoted

chemical modication to the dentin matrix but not

genipin when tested using µTBS.

With the addition of MMP inhibitors into

the adhesive system may improve the bond

strength and reduce the number of clinical steps.

However, this may have a negative impact on other

characteristics like degree of cure, elastic modulus,

water sorption, and solubility [45]. During the

incorporation process, consideration must be given

to the stability and sustainability of the additional

agents (MMP inhibitors) in the adhesive system.

The MMP inhibition capacity of the agent will

not be represented in the bond strength if non-

polymerizable agents without covalent bonding

capability are introduced to the adhesive, and

the only action that manifests is delaying but not

preventing the collagen degradation in the bond

interface [46]. Therefore, in the present study, the

natural MMP inhibitors were applied directly on

the etched dentin surface.

Fracture mode analysis revealed no

variation in adhesive or cohesive failure, among

all groups. After aging,

Moringa

5% showed

3 cohesive failures in resin composite, whereas

the control group had only adhesive failures.

The SEM analysis of the fractured fragments

indicated a shift in the weakest sites within

the resin dentin bond complex, aligning with

previous study [12]. Fractures were more likely

to occur in the adhesive layer or resin composite,

rather than dentin or at the bottom of a hybrid

layer after cross linker treatment. Although this

observation was not statistically signicant, it

suggests that the weakest site after cross linking

treatment was not within dentin.

In summary, the bond between resin and

dentin became more stable after bio modifying

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Anumula L et al.

Influence of two natural cr oss-linkers on microtensile bond str ength durability – An in vitro study

Anumula L et al.

Inﬂuence of two natural cross-linkers on microtensile bond

strength durability – An in vitro study

the dentin with 5% and 1%

Moringa

, even after

aging.

Centella

, on the other hand, did not

prove as effective in inhibiting MMP compared

Moringa

. Therefore, using a pretreatment

liner consisting of 5%

Moringa

can effectively

inhibit MMP and cross-link collagen, resulting in

improved bond strength. It is important to note

that this study was conducted in vitro, and further

research is necessary to evaluate the performance

of these natural agents in vivo conditions.

The findings of the study have important

implications for future research and clinical practice

in several ways. Firstly, they highlight the potential

of natural extracts as alternative MMP inhibitors,

for more safer and biocompatible adhesive systems.

Secondly, this study underscores the

importance of collagen cross linking in improving

the durability of resin dentin bonds.

Third, these findings encourage further

research into specific mechanisms by which

natural extracts exert their MMP inhibition and

collagen cross linking effects.

Author’s Contributions

LA: Conceptualization, Methodology,

Conceptualization, Methodology, Supervision,

Writing – Original Draft Preparation. SR:

Conceptualization, Formal Analysis, Resources,

Data Curation, Visualization. VSKK: Writing –

Review & Editing, Project Administration and

Funding Acquisition. RKA: Software, Validation,

Writing – Review & Editing. KG: Resources, Data

Curation, Investigation, Resources.

Conict of Interest

The authors have no proprietary, nancial,

or other personal interest of any nature or kind

in any product, service, and/or company that is

presented in this article.

Funding

This research did not receive any specic

grant from funding agencies in the public,

commercial or not-for-prot sectors.

Regulatory Statement

This study was conducted in accordance with

all the provisions of the local human subjects

oversight committee guidelines and policies

of institutional Ethical committee of Narayana

Dental College and Hospital. The approval code

for this study is IEC/NDCH, 2019/P-24.

REFERENCES

1. Villarroel M, Fahl N, De Sousa AM, De Oliveira OB Jr. Direct

esthetic restorations based on translucency and opacity of

composite resins. J Esthet Restor Dent. 2011;23(2):73-87.

http://dx.doi.org/10.1111/j.1708-8240.2010.00392.x. PMid:21477031.

2. Ravi R, Alla RK, Mohammed S, Devarhubli A. Dental composites -

a versatile restorative material: an overview. Indian J Dent Sci.

2013;5:111-5.

3. Swift EJ Jr, Perdigão J, Heymann HO. Bonding to enamel and

dentin: a brief history and state of the art, 1995. Quintessence

Int. 1995;26(2):95-110. PMid:7568728.

4. Pizzolotto L, Moraes RR. Resin composites in posterior teeth:

clinical performance and direct restorative techniques.

Dent J. 2022;10(12):222. http://dx.doi.org/10.3390/dj10120222.

PMid:36547038.

5. De Munck J, Van Landuyt K, Peumans M, Poitevin A, Lambrechts

P, Braem M,etal. A critical review of the durability of adhesion to

tooth tissue: methods and results. J Dent Res. 2005;84(2):118-32.

http://dx.doi.org/10.1177/154405910508400204. PMid:15668328.

6. Fonseca BM, Pleffken PR, Balducci I, Tay F, Pucci CR, Araujo MAM.

New trends in dentin bonding: treatment with chlorhexidine,

hyaluronic acid, vitaminc C and green tea. Braz Dent Sci.

2013;16(3):56-62. http://dx.doi.org/10.14295/bds.2013.v16i3.876.

7. Breschi L, Martin P, Mazzoni A, Nato F, Carrilho M, Tjäderhane

L,etal. Use of a specific MMP-inhibitor (galardin) for preservation

of hybrid layer. Dent Mater. 2010;26(6):571-8. http://dx.doi.

org/10.1016/j.dental.2010.02.007. PMid:20299089.

8. Guo X, Yu Y, Gao S, Zhang Z, Zhao H. Biodegradation of dental

resin-based composite: a potential factor affecting the bonding

effect: a narrative review. Biomedicines. 2022;10(9):2313.

http://dx.doi.org/10.3390/biomedicines10092313. PMid:36140414.

9. Tjäderhane L, Larjava H, Sorsa T, Uitto V-J, Larmas M, Salo T.

The activation and function of host matrix metalloproteinases

in dentin matrix breakdown in caries lesions. J Dent Res.

1998;77(8):1622-9. http://dx.doi.org/10.1177/0022034598077

0081001. PMid:9719036.

10. Uno S, Finger WJ. Function of the hybrid zone as a

stress-absorbing layer in resin-dentin bonding. Quintessence

Int. 1995;26(10):733-8. PMid:8935117.

11. Betancourt DE, Baldion PA, Castellanos JE. Resin-dentin

bonding interface: mechanisms of degradation and

strategies for stabilization of the hybrid layer. Int J Biomater.

2019;2019:e5268342. http://dx.doi.org/10.1155/2019/5268342.

PMid:30853990.

12. Tjäderhane L, Nascimento FD, Breschi L, Mazzoni A, Tersariol ILS,

Geraldeli S,etal. Strategies to prevent hydrolytic degradation

of the hybrid layer: a review. Dent Mater. 2013;29(10):999-1011.

http://dx.doi.org/10.1016/j.dental.2013.07.016. PMid:23953737.

13. Armstrong SR, Vargas MA, Chung I, Pashley DH, Campbell JA,

Laffoon JE, et al. Resin-dentin interfacial ultrastructure and

microtensile dentin bond strength after five-year water storage.

Oper Dent. 2004;29(6):705-12. PMid:15646228.

14. Anumula L, Ramesh S, Kolaparthi VSK, Kirubakaran R, Karobari MI,

Arora S,etal. Role of natural cross linkers in resin–dentin bond

durability: a systematic review and meta-analysis. Materials.

2022;15(16):5650. http://dx.doi.org/10.3390/ma15165650.

PMid:36013786.

15. Vidal CMP, Leme AA, Aguiar TR, Phansalkar R, Nam J-W, Bisson

J,etal. Mimicking the hierarchical functions of dentin collagen

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Anumula L et al.

Influence of two natural cr oss-linkers on microtensile bond str ength durability – An in vitro study

Anumula L et al.

Inﬂuence of two natural cross-linkers on microtensile bond

strength durability – An in vitro study

cross-links with plant derived phenols and phenolic acids.

Langmuir. 2014;30(49):14887-93. http://dx.doi.org/10.1021/

la5034383. PMid:25379878.

16. Aguiar TR, Vidal CMP, Phansalkar RS, Todorova I, Napolitano JG,

McAlpine JB,etal. Dentin biomodification potential depends on

polyphenol source. J Dent Res. 2014;93(4):417-22. http://dx.doi.

org/10.1177/0022034514523783. PMid:24574140.

17. Guedes MHG, Maia MHM, Graeff CFO, González AHM, Grecco

SS, D’Alpino P. Longevity of bonded composite restorations

after dentin biomodification with neolignans obtained from

Nectandra

leucantha

. Braz Dent Sci. 2022;25(3):e3300.

http://dx.doi.org/10.4322/bds.2022.e3300.

18. Al-Ammar A, Drummond JL, Bedran-Russo AK. The use of

collagen cross-linking agents to enhance dentin bond strength.

J Biomed Mater Res B Appl Biomater. 2009;91B(1):419-24.

http://dx.doi.org/10.1002/jbm.b.31417. PMid:19507140.

19. Bedran-Russo AKB, Pereira PNR, Duarte WR, Drummond JL,

Yamauchi M. Application of crosslinkers to dentin collagen

enhances the ultimate tensile strength. J Biomed Mater Res B

Appl Biomater. 2007;80B(1):268-72. http://dx.doi.org/10.1002/

jbm.b.30593. PMid:16767722.

20. Castellan CS, Bedran-Russo AK, Antunes A, Pereira PNR. Effect of

dentin biomodification using naturally derived collagen cross-linkers:

one-year bond strength study. Int J Dent. 2013;2013:918010.

http://dx.doi.org/10.1155/2013/918010. PMid:24069032.

21. Gough JE, Scotchford CA, Downes S. Cytotoxicity of glutaraldehyde

crosslinked collagen/poly(vinyl alcohol) films is by the

mechanism of apoptosis. J Biomed Mater Res. 2002;61(1):121-30.

http://dx.doi.org/10.1002/jbm.10145. PMid:12001254.

22.

Porto ICCM, Nascimento TG, Oliveira JMS, Freitas PH, Haimeur A,

França R. Use of polyphenols as a strategy to prevent bond degradation

in the dentin-resin interface. Eur J Oral Sci. 2018;126(2):146-58.

http://dx.doi.org/10.1111/eos.12403. PMid:29380895.

23. Merecz-Sadowska A, Sitarek P, Kucharska E, Kowalczyk T, Zajdel

K, Cegliński T,et al. Antioxidant properties of plant-derived

phenolic compounds and their effect on skin fibroblast cells.

Antioxidants. 2021;10(5):726. http://dx.doi.org/10.3390/

antiox10050726. PMid:34063059.

24. Van Meerbeek B, Peumans M, Poitevin A, Mine A, Van Ende A,

Neves A,etal. Relationship between bond-strength tests and

clinical outcomes. Dent Mater. 2010;26(2):e100-21. http://dx.doi.

org/10.1016/j.dental.2009.11.148. PMid:20006379.

25. Amaral FLB, Colucci V, Palma-Dibb RG, Corona SAM.

Assessment of in vitro methods used to promote adhesive

interface degradation: a critical review. J Esthet Restor Dent.

2007;19(6):340-53, discussion 354. http://dx.doi.org/10.1111/

j.1708-8240.2007.00134.x. PMid:18005284.

26. Wagner H, Bladt S, Zgainski EM. Plant drug analysis: a thin

layer chromatography atlas. Berlin: Springer-Verlag; 1984.

http://dx.doi.org/10.1007/978-3-662-02398-3.

27. Cai J, Palamara JEA, Burrow MF. Effects of collagen crosslinkers on

dentine: a literature review. Calcif Tissue Int. 2018;102(3):265-79.

http://dx.doi.org/10.1007/s00223-017-0343-7. PMid:29058055.

28. Chung L, Dinakarpandian D, Yoshida N, Lauer-Fields JL, Fields

GB, Visse R,etal. Collagenase unwinds triple-helical collagen

prior to peptide bond hydrolysis. EMBO J. 2004;23(15):3020-30.

http://dx.doi.org/10.1038/sj.emboj.7600318. PMid:15257288.

29. Hiraishi N, Sono R, Sofiqul I, Yiu C, Nakamura H, Otsuki M,etal.

In vitro evaluation of plant-derived agents to preserve dentin

collagen. Dent Mater. 2013;29(10):1048-54. http://dx.doi.

org/10.1016/j.dental.2013.07.015. PMid:23942145.

30. Islam MS, Hiraishi N, Nassar M, Yiu C, Otsuki M, Tagami J.

Effect of hesperidin incorporation into a self-etching primer

on durability of dentin bond. Dent Mater. 2014;30(11):1205-12.

http://dx.doi.org/10.1016/j.dental.2014.08.371. PMid:25194169.

31.

Leopoldini M, Russo N, Toscano M. The molecular basis of working

mechanism of natural polyphenolic antioxidants. Food Chem.

2011;125(2):288-306. http://dx.doi.org/10.1016/j.foodchem.2010.08.012.

32. Brglez Mojzer E, Knez Hrnčič M, Škerget M, Knez Ž, Bren U.

Polyphenols: extraction methods, antioxidative action, bioavailability

and anticarcinogenic effects. Molecules. 2016;21(7):901.

http://dx.doi.org/10.3390/molecules21070901. PMid:27409600.

33. Lim H, Kim HP. Inhibition of mammalian collagenase,

matrix metalloproteinase-1, by naturally-occurring

flavonoids. Planta Med. 2007;73(12):1267-74. http://dx.doi.

org/10.1055/s-2007-990220. PMid:17886198.

34. Rosa WLO, Piva E, Silva AF. Bond strength of universal adhesives: a

systematic review and meta-analysis. J Dent. 2015;43(7):765-76.

http://dx.doi.org/10.1016/j.jdent.2015.04.003. PMid:25882585.

35. Yaghmoor RB, Jamal H, Abed H, Allan E, Ashley P, Young A.

Incorporation of MMP inhibitors into dental adhesive systems

and bond strength of coronal composite restorations: a

systematic review and meta-analysis of in vitro studies. Jpn

Dent Sci Rev. 2022;58:298-315. http://dx.doi.org/10.1016/j.

jdsr.2022.09.004. PMid:36247748.

36. Carrilho MRO, Carvalho RM, Tay FR, Pashley DH. Effects of

storage media on mechanical properties of adhesive systems.

Am J Dent. 2004;17(2):104-8. PMid:15151336.

37. Örtengren U, Andersson F, Elgh U, Terselius B, Karlsson S. Influence

of pH and storage time on the sorption and solubility behaviour of

three composite resin materials. J Dent. 2001;29(1):35-41. http://

dx.doi.org/10.1016/S0300-5712(00)00055-5. PMid:11137637.

38. Hashimoto M, Ohno H, Sano H, Tay FR, Kaga M, Kudou Y,etal.

Micromorphological changes in resin-dentin bonds after 1 year

of water storage. J Biomed Mater Res. 2002;63(3):306-11.

http://dx.doi.org/10.1002/jbm.10208. PMid:12115762.

39. Armstrong SR, Keller JC, Boyer DB. The influence of water storage

and C-factor on the dentin-resin composite microtensile bond

strength and debond pathway utilizing a filled and unfilled

adhesive resin. Dent Mater. 2001;17(3):268-76. http://dx.doi.

org/10.1016/S0109-5641(00)00081-6. PMid:11257301.

40. Aguilar LT, Rezende NPM, Reis A, Loguercio AD, Grande

RHM, Ballester RY,etal. Tensile bond strength of adhesive

systems--effects of primer and thermocycling. Pesqui Odontol

Bras. 2002;16(1):37-42. http://dx.doi.org/10.1590/S1517-

74912002000100007. PMid:11938716.

41.

Gale MS, Darvell BW. Thermal cycling procedures for laboratory

testing of dental restorations. J Dent. 1999;27(2):89-99.

http://dx.doi.org/10.1016/S0300-5712(98)00037-2. PMid:10071465.

42.

Saffarpour A, Valizadeh S, Amini A, Kharazifard M-J, Rohaninasab M.

Effect of matrix metalloproteinase inhibitors on microtensile bond

strength of dental composite restorations to dentin in use of an

etch-and-rinse adhesive system. Clin Exp Dent Res. 2020;6(6):686-92.

http://dx.doi.org/10.1002/cre2.313. PMid:32989895.

43. Zheng P, Zaruba M, Attin T, Wiegand A. Effect of different matrix

metalloproteinase inhibitors on microtensile bond strength of

an etch-and-rinse and a self-etching adhesive to dentin. Oper

Dent. 2015;40(1):80-6. http://dx.doi.org/10.2341/13-162-L.

PMid:24815915.

44. Nagpal M, Taneja S, Kumar M, Dudeja C. Evaluation of the effect

of natural versus synthetic matrix metalloproteinase silencers

in preservation of dentin collagen and long-term bond strength

of total etch adhesive. J Conserv Dent. 2020;23(3):221-6.

http://dx.doi.org/10.4103/JCD.JCD_91_20. PMid:33551589.

45. Simmer FS, Silva EM, Bezerra RSG, Miranda MESNG, Noronha JD

Fo, Amaral CM. Bond stability of conventional adhesive system

with MMP inhibitors to superficial and deep dentin. J Mech Behav

Biomed Mater. 2019;100:103402. http://dx.doi.org/10.1016/j.

jmbbm.2019.103402. PMid:31493688.

46. Pashley DH, Tay FR, Imazato S. How to increase the durability of

resin-dentin bonds. Compend Contin Educ Dent. 2011;32(7):60-4,

66. PMid:21910364.

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3869

Anumula L et al.

Influence of two natural cr oss-linkers on microtensile bond str ength durability – An in vitro study

Anumula L et al.

Inﬂuence of two natural cross-linkers on microtensile bond

strength durability – An in vitro study

Lavanya Anumula

(Corresponding address)

Narayana Dental College and Hospital, Department of Conservative Dentistry and

Endodontics, Chintareddypalem, Nellore, A.P. India.

Email: lavanyamds@gmail.com

Date submitted: 2023 Apr 28

Accept submission: 2023 July 18