Long-term effect of ammonia- and water-based silver fluoride on dentin 
collagen matrix
Merve Uctasli a,* , Roda Seseogullari-Dirihan a,b , Thiago Henrique Scarabello Stape a,c ,  
Mustafa Murat Mutluay a,d , Arzu Tezvergil-Mutluay a,c
a Department of Restorative Dentistry and Cariology, Adhesive Dentistry Research Group, Institute of Dentistry, University of Turku, Turku, Finland
b Department of General Dental Science, School of Dentistry, Marquette University, Milwaukee, WI, USA
c Turku University Hospital, TYKS, University of Turku, Turku, Finland
d Department of Oral and Maxillofacial Diseases, Faculty of Medicine, University of Helsinki, Helsinki, Finland
A R T I C L E  I N F O
Keywords:
Dentin
Silver diamine fluoride (SDF)
Aqueous silver fluoride (SF)
Potassium iodide (KI)
Proteases
Degradation
Inactivation
Matrix metalloproteinases
Cysteine cathepsins
Dentin collagen
A B S T R A C T
Objective: To evaluate the long-term effect of ammonia- and water-based silver fluoride treatments on the 
degradation of the dentin collagen matrix.
Methods: Dentin beams (0.3x3x7mm) were demineralized (10 % H3PO4), rinsed and randomly distributed into six 
groups. Groups (n ˆ 10 beams/group) were treated with (1) ammonia-based silver fluoride ˆ SDF; (2) SDF 
‡ potassium iodide ˆ KI (3) water-based silver fluoride ˆ SF (4) SF ‡ KI (5) KI (6) untreated demineralized 
dentin beams served as control. Following treatments, dry mass, modulus of elasticity and enzymatic activity 
were assessed. Dentin beams were incubated in calcium- and zinc-containing artificial saliva up to 6 months. 
After different incubation periods (1 week, 1 month, 3 months or 6 months), dry mass, modulus of elasticity and 
enzymatic activity were reevaluated. The aliquots of incubation media were analyzed to determine the solubi-
lized telopeptides of collagen (ICTP and CTX immunoassays), hydroxyproline release and total extractable 
protein (Bradford assay). Scanning electron microscopy imaging and in situ zymography analyses were con-
ducted. Data were statistically analyzed with ANOVA followed by Tukey test (αˆ0.05).
Results: Silver fluoride treatments reduced the total enzymatic activity, but increased the solubilized telopeptides 
of collagen throughout incubation periods (p < 0.05). The addition of KI exacerbated the loss of dry mass, 
modulus of elasticity, hydroxyproline release and total protein loss (p < 0.05).
Significance: Ammonia- and water-based silver fluoride treatments may reduce long-term degradation of dentin 
collagen. However, potassium iodide can further increase endogenous protease activity and compromise the 
structural integrity of dentin’s organic matrix.
1. Introduction
Dentin caries is a multifactorial, dynamic and the most prevalent oral 
disease that still constitutes a significant public health challenge and 
persists as a global health burden [1,2]. The progression of dentin caries 
involves the demineralization of hydroxyapatite and degradation of 
dentin collagen matrix. These processes are driven by bacterial acid 
production and enzymatic biodegradation which together lead to the 
demineralization of dentin and the weaken of the collagen matrix, 
respectively [3,4]. Dentin matrices contain proteolytic enzymes such as 
matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs). These 
enzymes are trapped in mineralized dentin. Exposure of dentin to low 
pH during cariogenic activity or the acid etching step of adhesive pro-
cedures [5], can activate endogenous enzymes, leading to degradation 
of dentin collagen matrix and decreased restoration durability [3,6,7]. 
Therefore, inhibition of endogenous activity is critical for slowing caries 
progression and decreasing the bond degradation, in other words, 
reducing collagen degradation at resin-dentin bonding interfaces over 
time [4,6,8]. Strategies including the use of collagen crosslinkers, 
enzyme inhibitors, remineralization agents, selective or non-selective 
inhibitors have been studied to prevent degradation of dentin collagen 
matrix by enzymes.
* Correspondence to: Institute of Dentistry, University of Turku, Lemminkaisenkatu 2, Turku 20520, Finland.
E-mail addresses: meucta@utu.fi (M. Uctasli), roda.seseogullaridirihan@marquette.edu (R. Seseogullari-Dirihan), thiago.stape@utu.fi (T.H.S. Stape), mmutluay@ 
utu.fi (M.M. Mutluay), arztez@utu.fi (A. Tezvergil-Mutluay). 
Contents lists available at ScienceDirect
Dental Materials
journal homepage: www.elsevier.com/locate/dental
https://doi.org/10.1016/j.dental.2025.10.016
Received 18 July 2025; Received in revised form 24 October 2025; Accepted 28 October 2025  
Dental Materials xxx (xxxx) xxx 
0109-5641/© 2025 The Author(s). Published by Elsevier Inc. on behalf of The Academy of Dental Materials. This is an open access article under the CC BY license 
( http://creativecommons.org/licenses/by/4.0/ ). 
Please cite this article as: Merve Uctasli et al., Dental Materials, https://doi.org/10.1016/j.dental.2025.10.016 
The contemporary philosophy of caries management has shifted 
from the traditional drill and fill approach to a seal and heal model, 
emphasizing preventive strategies such as fluoride therapy [9]. A variety 
of fluoride-containing agents have been used in dentistry to control the 
progression of dentin caries and promote remineralization. Combining 
fluoride with silver, an antimicrobial and anticariogenic agent, silver 
fluoride-based treatments are formed. Silver fluoride-based treatments 
have two different chemical formulas depending on the third ion 
included. When silver and fluoride ions form a complex with ammonia, 
ammonia-based silver fluoride, known as silver diamine fluoride (SDF), 
is formed [10]. Recently, a water-based silver fluoride, in other words, 
an aqueous silver fluoride (SF), has been introduced as the first 
water-based silver fluoride solution. In this formulation, ammonia ion is 
replaced by water and provided patient benefits such as improved smell, 
taste and reduced soft tissue irritation. However, both formulations raise 
esthetic concerns due to silver staining of tooth and surrounding tissues, 
caused by oxidation of free silver ions [11], which can limit their 
acceptance in clinical practice. To address this issue, the application of 
potassium iodide (KI) immediately after treatment with either ammonia- 
or water-based silver fluoride leads to the formation of a white silver 
iodide compound that ameliorates black dentin stains [12]. Therefore, 
silver fluoride treatments can be achieved either ammonia- and 
water-based silver fluoride alone (SDF & SF) or in combination with 
potassium iodide (KI) application.
The use of ammonia-based SDF has gained popularity due to possible 
inhibition of endogenous proteases [13,14]. It has also emerged as a 
possible off-label approach to extend the longevity of resin-dentin in-
terfaces [8]. Furthermore, according to the World Health Organization 
(WHO) Global Strategy Action Plan [15], by 2030, 50 % of countries 
should include SDF in their list of essential medicines. This highlights 
the growing potential and increased use of the SDF in preventive and 
restorative dentistry. Although antimicrobial benefits of SDF are well 
known on caries arrest, the effect of SF and the combinations with KI 
treatment require further investigation. Limited number of studies have 
evaluated the short-term effects of ammonia-based silver fluoride (SDF) 
[13,14] and water-based silver fluoride (SF) [16] on dentin endogenous 
activity. To the best of our knowledge, the long-term influence of 
ammonia- and water-based silver fluoride treatments on dentin endog-
enous enzymatic activity has yet to be investigated. To date, it remains 
unclear whether ammonia- and water-based silver fluoride treatments 
can reduce or inactivate endogenous dentin enzymatic activity over 
extended periods.
The aim of this study was to evaluate the long-term effect of 
ammonia- and water-based silver fluoride treatments on their ability to 
inactivate dentin proteases and prevent dentin matrix degradation. The 
inactivation of dentin MMPs and CCs were assessed by means of quan-
titation of ICTP (crosslinked carboxyterminal telopeptide of type I 
collagen) and CTX (C-terminal crosslinked telopeptide of type I 
collagen) release, respectively. Furthermore, dentin matrix degradation 
were evaluated by measuring loss of dry mass, changes in the modulus of 
elasticity, total enzymatic activity, hydroxyproline release and total 
extractable protein. To support the findings, imaging techniques, scan-
ning electron microscopy (SEM) and in situ zymography were used. The 
null hypotheses tested were ammonia- and water-based silver fluoride 
treatments applied on demineralized dentin matrix do not affect the 
degradation of collagen matrix or dentin protease activity.
2. Materials and methods
Sixty intact third molars extracted during routine dental treatments 
from anonymous donors were used in the study and exempt from ethical 
notification in accordance with local regulations (Tissue act, section 20). 
Teeth were stored at 4 C in 0.9 % NaCl supplemented with 0.02 % NaN3 
to prevent bacterial growth and used within three months of extraction.
The sample size was determined using G*Power (version 3.1.9.7, 
Kiel University, Kiel, Germany) based on the results of a prior pilot study 
[17]. The effect size (f) was 0.40, the type I error (α) was 0.05 and the 
statistical power (1-β) was 0.95. The total sample size required for this 
study was 48, with a minimum sample size of 8 specimens per group. In 
the present study 10 specimens per group were used.
2.1. Preparation of dentin beam specimens
During the preparation of test specimens, a low-speed water-cooled 
saw (Isomet 1000 Precision Saw, Buehler Ltd., USA) were used. One 
dentin disc from the mid-coronal dentin region of each tooth (n ˆ 10/ 
group) with the thickness of 0.3 (0.02) mm was obtained by sectioning 
perpendicularly to the tooth’s long axis. The dentin discs were then 
sectioned mesio-distally to produce rectangular dentin beams with the 
dimensions of 0.3 mm thickness x 3 mm width x 7 mm length. To enable 
repeated measurements to be performed on the same surface, a dimple 
was made at the corner of each dentin beam on the occlusal surface. 
Prepared dentin beams were stirred in aqueous 10 wt% H3PO4 (Phos-
phoric acid, Merck Sigma-Aldrich, Germany) for 40 min at 25 C for 
complete demineralization and then rinsed in distilled water at 4 C for 
1 h under constant stirring at 60 rpm (Loopster digital, IKA, Germany). 
Digital radiography was used to confirm the absence of residual minerals 
[18]. After demineralization process, digital images were obtained on a 
stereo microscope (Leica M60, Leica Microsystems, Germany) and 
thickness and width dimension of the demineralized beams were pre-
cisely measured using an open-source image software (ImageJ, National 
Institute of Health, USA). Then, dentin beams were placed in individu-
ally labelled 96-well plates. Initial modulus of elasticity of each beam 
was measured with three point bending test using universal testing 
machine (Autograph AGS-X Series, Shimadzu, Japan), then beams were 
dried in vacuum desiccator containing dry silica beads for 72 h and 
initial dry mass of each beam was weighted. After initial baseline 
measurements of modulus of elasticity and dry mass, demineralized 
dentin beams (n ˆ 10/group) were assigned into 6 balanced groups, so 
that their mean modulus of elasticity and dry mass values were statis-
tically similar. Commercially available ammonia- and water-based silver 
fluoride treatments (Riva Star and Riva Star Aqua, SDI, Australia) were 
selected for this study and the pH values of each solution was measured 
with pH meter (PHM210, Radiometer Analytical, France) as shown in 
Table 1.
Groups were prepared according to the silver fluoride treatments 
received (Table 1) and demineralized dentin beams without any silver 
fluoride treatment were served as control. Group 1: Silver Diamine 
Fluoride ˆ SDF (Riva Star Bottle 1, SDI, Australia); Group 2: SDF 
‡ Potassium Iodide ˆ KI (Riva Star Bottle 1 and 2, SDI, Australia); Group 
3: Aqueous silver fluoride ˆ SF (Riva Star Aqua Bottle 1, SDI, Australia); 
Group 4: SF ‡ KI (Riva Star Aqua Bottle 1 and 2, SDI, Australia); Group 
5: KI (Riva Star Aqua Bottle 2, SDI, Australia); Group 6: demineralized 
dentin beams without any silver fluoride treatment as control group. 
Demineralized dentin beams in test groups 1–5 were treated with cor-
responding silver fluoride treatments for 1 min according to the manu-
facturer`s instructions (Table 1). After silver fluoride treatments, beams 
were washed with distilled water inside 96-well plate at a microplate 
shaker at 300 rpm, then beams were blot dried with absorbent paper. 
Total enzymatic activity (generic MMP assay), modulus of elasticity and 
dry mass of all dentin beams were measured. Then, each dentin beam 
was placed into individually labeled O-ring polypropylene tube con-
taining 500 µl artificial saliva (pH 7.4) containing 5 mM HEPES, 2.5 mM 
CaCl2
. H2O, 0.05 mM ZnCl2, and 0.3 mM NaN3 [19] and incubated in a 
shaking-water-bath (60 cycles/min) at 37 C, to facilitate artificial saliva 
diffusion within collagen fibrils, for designated incubation periods of 1 
week, 1 month, 3 months and 6 months. After each incubation period, 
the incubation medium was stored frozen (-80 C) until the end of the 
experiment. Those media were analyzed for solubilized telopeptides of 
collagen (ICTP & CTX), hydroxyproline (HYP) release and total 
extractable protein (Bradford assay).
M. Uctasli et al.                                                                                                                                                                                                                                 Dental Materials xxx (xxxx) xxx 
2 
2.2. Loss of dry mass
The loss of demineralized dry dentin mass was used as an indirect 
measurement of the enzymatic degradation of dentin matrix as a result 
of total endogenous protease activity as previously described [20,21]. 
Briefly, dentin beams measuring 0.3  37 mm (n ˆ 10/group) were 
demineralized in aqueous 10 wt% H3PO4 for 40 min, rinsed in distilled 
water for 1 h, transferred into individually labelled 96-well plates and 
placed in a vacuum desiccator for 72 h [18]. Specimens were then 
weighed individually with an analytical microbalance (XP6 Microbal-
ance, Mettler Toledo, USA). After the initial dry mass measurements, 
dentin beams were re-hydrated in distilled water at 4 C for 1 h. Ac-
cording to previous findings, such rehydration period is sufficient for 
complete re-expansion of the dried dentin beams [22]. After hydration, 
silver fluoride treatments were applied (Table 1), demineralized dentin 
beams without any silver fluoride treatment served as control. The 
measurement of dry mass loss was reassessed immediately after silver 
fluoride treatments following the same protocol. After each incubation 
period, dentin beams were rinsed at 4 C for 24 h in distilled water to 
remove media salts before dry mass measurements. Calculation of the 
dry mass was repeated under the same conditions after dehydration in 
desiccator. The loss of dry mass was calculated as the percentage change 
of dry mass loss referring to the dry mass recorded for each beam after 
corresponding silver fluoride treatments. Demineralized dentin beams 
without any treatment served as control group.
2.3. Modulus of elasticity
The 3-point bending test was selected to investigate the modulus of 
elasticity since it allows repeated measurements to be performed 
nondestructively [23]. Dentin beams were placed on a 3-point bending 
jig (Instron, Instron Inc., USA) with a 5 mm span length between sup-
ports, fully immersed in distilled water and tested under flexure to 3 % 
strain at a displacement rate of 0.5 mm min 1 using a 5 N load cell 
(SMT1–5N, Interface, USA) on a universal testing machine (Autograph 
AGS-X, Shimadzu, Japan). The load was immediately returned to 0 % 
stress within 15 s after maximum displacement to prevent creep of the 
demineralized collagen matrix [20,21]. Load-displacement curves were 
converted to stress-strain curves and the apparent modulus of elasticity 
(E) were then calculated using the following equation and expressed in 
MPa: E ˆ mL3/4bh3, where m is the slope of the linear portion of the 
load-displacement curve (N/mm), L is the span length (5 mm), b is the 
width and h is the thickness of demineralized dentin beam in mm. Each 
demineralized dentin beam was tested in duplicates and the average was 
used as the E value of each demineralized dentin beam. E were evaluated 
initially on demineralized dentin beams which served as baseline mea-
surements, after the application of silver fluoride treatments (Table 1), 
and after each designated incubation period (1 week, 1 month, 3 months 
or 6 months). Demineralized dentin beams without any treatment 
served as control.
2.4. Total enzymatic activity
A generic colorimetric MMP assay (Sensolyte Generic MMP assay, 
Anaspec, USA) was used to determine whether the ammonia- and water- 
based silver fluoride treatments could inactivate the dentin-derived 
endogenous total MMP activity [24]. Demineralized dentin beams 
were used as the MMP source [21]. After silver fluoride treatments 
(Table 1), dentin beams were rinsed, blot-dried with absorbent paper 
and were incubated in 200 μl of the substrate and assay buffer in the 
96-well plate for 2 h at 25 ºC to assess the MMP activity. Demineralized 
dentin beams without any treatment served as control. Every 30 min, 
dentin beams were removed from the wells and the MMP activity [25]
was measured using a spectrometer (Synergy HT, BioTek Inst, USA) at 
412 nm wavelength. Data were recorded after each measurement until 
dentin beam activity reached the peak value. Then, beams were rinsed 
free of MMP substrate in distilled water and incubated at designated 
incubation periods (1 week, 1 month, 3 months or 6 months). After each 
incubation period, beams were rinsed and the total enzymatic activity 
was reassessed following the same protocol. The percentage inhibition 
of MMPs were calculated by subtracting the mean values of the control 
group (demineralized dentin beams without any silver fluoride treat-
ment) from ammonia- and water-based silver fluoride treatment groups, 
then dividing by the mean value of the control group.
2.5. Solubilized telopeptides of collagen
Release of different C-telopeptide fragments were analyzed from the 
incubation media after each designated incubation period (1 week, 1 
month, 3 months or, 6 months) to evaluate the specific role of MMPs and 
CCs in type I collagen degradation. Matrix degradation by MMPs were 
determined by measuring the amount of solubilized type I collagen C- 
terminal cross-linked telopeptides [26], cathepsin K-induced degrada-
tion of type I collagen were measured by the amount of solubilized 
Table 1 
Materials, compositions, pH values and application procedures of the ammonia- and water-based silver fluoride treatments.
Abbreviation Material Composition pH* Application Lot 
number
Ammonia- 
based 
silver 
fluoride
​ Riva Star (SDI, 
Australia)
​ ​ 1211902
SDF Bottle 1: Silver 
Diamine Fluoride
silver (32.6 % w/v), 
fluoride (5.7 % w/ 
v), 
ammonia (15–20 % 
w/v), 
water (40–60 % w/ 
v)
12.05 Active application of 2 µl on each dentin beam surface for 60 s. 1210634
KI Bottle 2: Potassium 
Iodide
potassium iodide  
(58 % w/w)
8.63 Active application of 4 µl on each dentin beam surface for 60 s until the 
initially creamy white appearance turns clear.
1211767
Water-based 
silver 
fluoride
​ Riva Star Aqua (SDI, 
Australia)
​ ​ 1204201
SF Bottle 1: Aqueous 
Silver Fluoride
silver (32.6 % w/v), 
fluoride (5.7 % w/ 
v), 
water (50–70 % w/ 
v)
7.73 Active application of 2 µl on each dentin beam surface for 60 s. 221050
KI Bottle 2: Potassium 
Iodide
potassium iodide  
(58 % w/w)
7.94 Active application of 4 µl on each dentin beam surface for 60 s until the 
initially creamy white appearance turns clear.
221048
* The pH values of tested materials were determined at Adhesive Dentistry Research Laboratory, Institute of Dentistry, University of Turku, Turku, Finland.
M. Uctasli et al.                                                                                                                                                                                                                                 Dental Materials xxx (xxxx) xxx 
3 
C-terminal peptide, using the commercially available kits ICTP (UniQ 
ICTP RIA, Aidian Diagnostics, Finland) and CTX (Serum Crosslaps 
ELISA, IDS, Denmark), respectively. The only known source of CTX is 
cathepsin K [27]. Following each incubation period (1 week, 1 month, 3 
months or 6 months), 20–25 μl aliquots of incubation media from each 
tube were used to quantitate solubilized ICTP and CTX fragments 
following the kits protocol. The measurements was performed using a 
gamma counter (Wallac Wizard, Turku, Finland) at 1000 counts for ICTP 
and a spectrophotometer (Synergy HT, Bio Tek Inst., USA) at 450 nm 
absorbance, respectively. The amount of collagen degradation as ICTP 
and CTX telopeptide fragment release was calculated with a standard 
curve using standards with known concentrations provided by the 
manufacturer.
2.6. Hydroxyproline quantification
The amount of collagen solubilization was assessed by hydroxypro-
line (HYP) quantification from the incubation media after each desig-
nated incubation period (1 week, 1 month, 3 months or 6 months). 
Solubilized collagen peptide fragments were assessed following a pre-
viously described HYP quantification protocol [28]. Briefly, aliquots of 
HYP standards (2–20 µg) prepared from stock solutions and 25 µl of 
incubation media were mixed with 25 µl of 4 N NaOH (2 N final con-
centration) in a total volume of 50 µl in 2 mL Nalgene O-ring tubes. 
Specimens were autoclaved at 120 C for 20 min. 450 µl of chloramine-T 
was added to hydrolyzed tubes and mixed gently to allow oxidation for 
20 min at room temperature. 500 µL Ehrlich’s aldehyde reagent was 
added to each specimen for chromophore formation by incubating the 
specimens at 65 C for 20 min. Absorbance values were obtained using a 
spectrophotometer (Model UV-A180, Shimadzu, Japan) at 550 nm and 
plotted against the standard HYP curves to determine the HYP release.
2.7. Total extractable protein
Extracted dentin proteins obtained from demineralized dentin were 
used for the quantification of extractable protein level from the incu-
bation media after each designated incubation period (1 week, 1 month, 
3 months or 6 months). Extracted proteins from each dentin beam into 
the incubation media were tested according to the manufacturer’s in-
structions using a commercial kit based on the Bradford assay (Bio-Rad, 
Hercules, USA) [29]. Briefly, 1–50 μg/mL series of bovine serum albu-
min (BSA) standards (50 μl), and aliquots of 50 μl of incubation media 
were analyzed. After 5 min of incubation to allow for color development, 
the absorbance was measured using a spectrophotometer (Synergy HT, 
BioTek Inst, USA) at 595 nm. A linear standard curve was prepared by 
plotting the average reading for each BSA standard vs. its concentration 
in μg/mL and the standard curve was used to determine the total 
extractable protein concentration.
2.8. Scanning electron microscopy (SEM)
Additional 20 dentin beams were prepared, demineralized and 
treated with ammonia- and water-based silver fluoride treatments 
(Group 1–4) following the according to the manufacturer instructions 
(Table 1), demineralized dentin beams without any silver fluoride 
treatment served as control group (n ˆ 4/group). Group 5, where only KI 
was applied on dentin beams was not prepared for SEM analysis, 
because KI application alone is not used in clinical practice. Half of the 
specimens were evaluated for the surface view of the dentine after silver 
fluoride treatments, the other half were fractured using liquid nitrogen 
to obtain a cross-sectional view. Specimens were dehydrated in 
ascending series of ethanol (25, 50, 75, 95 and 100 %), fixed in HMDS 
(Sigma Aldrich, USA) [30], mounted on aluminum stubs, sputter coated 
with gold-palladium and analyzed by SEM (Phenom ProX, 
Phenom-World, USA) on backscattering mode at 10 kV up to 
2500  magnifications.
2.9. Evaluation of gelatinase activity by in situ zymography
Ten teeth were coronally sectioned under water-cooling to expose 
flat midcoronal dentin surfaces using low speed diamond saw (Isomet 
1000 Precision Saw, Buehler Ltd, USA). Absence of remaining enamel 
was verified with a stereo microscope (Leica M60, Leica Microsystems, 
Germany) at 40  magnification. Smear layer standardization was 
performed by wet-polishing exposed dentin surfaces with 320-grit sili-
con carbide (SiC) paper (CarbiMet, Buehler Ltd, USA) for 60 s at 350 rpm 
(MetaServ 250 Grinder-Polish, Buehler Ltd, USA). Bonding was followed 
the etch-and-rinse protocol, where dentin surfaces were etched with 
37 % phosphoric acid gel (Scotchbond Universal Etchant, Solventum, 
USA) for 5 s, rinsed with water for 15 s and blot dried with absorbent 
paper. Ammonia- and water-based silver fluoride treatments (Group 
1–4) were applied as in the SEM analysis according to the manufacturer 
instructions (Table 1) (n ˆ 2/group) after etching step, before adhesive 
application. The etched dentin without any silver fluoride treatment 
served as control group. Group 5, where only KI was applied on dentin 
beams was not prepared for in situ zymography evaluation as KI appli-
cation alone is not used in clinical practice. Active adhesive application 
(Scotchbond Universal Plus, Solventum, USA) for 20 s, gentle solvent 
evaporation for 10 s and light curing with a LED light curing unit (Valo 
Corded, Ultradent, USA) at 1800 mW/cm2 for 10 s were achieved. 
Composite blocks were built with a nanofilled composite resin (Filtek 
Ultimate Universal Restorative, Solventum, USA) in one 2-mm incre-
ment and was light cured for 20 s. All bonding procedures were carried 
out by a single operator.
Bonded specimens were then cut vertically into 0.3 mm thick slabs to 
expose adhesive/dentin interfaces, wet-polished with 600, 1200 and 
2000 SiC paper and ultrasonically cleaned for 5 min after the polishing 
steps. The four middle specimens were selected for evaluation. 0.3 mm- 
thick sections were demineralized with 10 % phosphoric acid for 10 s 
and rinsed with distilled water for 20 s following blot-drying with 
absorbent paper. A self-quenched fluorescein-conjugated gelatin (E- 
12055, Molecular Probes, Eugene, USA) from a stock solution of DQ- 
gelatin (DQ-gelatin, E12055; Molecular Probes, Eugene, USA) were 
diluted 1:8 in the dilution buffer (NaCl 150 mM, CaCl2 5 mM, Tris-HCl 
50 mM, pH 8.0) as described previously [31]. Specimens were wetted 
with 30 μl of gelatin solution with and without fluorescent gel, to pre-
vent false readings, and then covered with a coverslip. The slides were 
light protected and incubated in humified chamber at 37 C for 24 h, 
interfacial endogenous gelatinase activity were observed using a 
confocal laser scanning microscope (Zeiss LSM 880, Carl Zeiss, Ger-
many) at 488 nm for excitation wavelength. The entire resin-dentin 
interface was examined and sequential images (z-stack) of the bonded 
interface were recorded from each dentin slice. Then, images were 
quantitatively assessed using an open-source image software (ImageJ, 
National Institute of Health, USA) by a single-blinded examiner. Read-
ings obtained with the gelatin solution, without fluorescent gel, were 
used as background readings. The gelatinolytic activity was expressed as 
the percentage of the fluorescence area.
2.10. Statistical analysis
The percent loss of dry mass, modulus of elasticity, total enzymatic 
activity, total endogenous activity, hydroxyproline quantification, total 
extractable protein and in situ zymography data were compared for 
normality (Shaphiro-Wilk test) and homoscedasticity (Levene test). The 
normality and equality variance assumptions of the data were valid. In 
situ zymography data were analyzed using one-way ANOVA. Remaining 
data were analyzed using two-way repeated measures of ANOVA. Post- 
hoc multiple comparisons were performed with the Tukey test. Signifi-
cance level (α) of 0.05 was used for all statistical tests. Statistical ana-
lyses were performed using IBM SPSS Statistics for Windows, version 28 
(IBM Corp, USA).
M. Uctasli et al.                                                                                                                                                                                                                                 Dental Materials xxx (xxxx) xxx 
4 
3. Results
3.1. Loss of dry mass
Fig. 1 shows the cumulative loss of dry mass for demineralized dentin 
beams among all groups and incubation periods. Untreated demineral-
ized dentin beams served as control group, which showed a significant 
increase in dry mass loss after 1 months (p < 0.05). Treatment with SDF 
alone exhibited significantly lower dry mass loss at all incubation pe-
riods compared to the control (p < 0.05), without any significant dif-
ferences within the group (p > 0.05). SF did not significantly differ from 
the control group at 1-week incubation (p > 0.05), however, SF treat-
ments resulted in reduced dry mass loss compared to the control beyond 
1 week incubation (p < 0.05). There were no significant differences 
within the SF group at different incubation periods (p > 0.05). The 
additional treatment of KI after either SDF or SF significantly increased 
the dry mass loss compared to their respective single treatments and the 
control (p < 0.05). The SDF ‡ KI treatment (23.8  4.9 %) showed a 
higher dry mass loss compared to the SF ‡ KI group (11.1  2.2 %) 
(p < 0.05). Both groups displayed significant differences in dry mass loss 
from 1 week to 6 months incubation (p < 0.05). SF ‡ KI treatment 
revealed similar results with the control group at 3 and 6 months in-
cubation (p > 0.05). Treatment with KI alone resulted in the highest dry 
mass loss among all groups (p < 0.05) yet exhibited no significant 
change over different incubation periods (p > 0.05).
3.2. Modulus of elasticity
The average elastic modulus of demineralized dentin collagen for all 
specimens at baseline (n ˆ 60) was 7.28  1.23 MPa. Changes in elastic 
modulus across all groups and incubation periods are shown in Fig. 2. 
Untreated demineralized dentin beams served as control group and did 
not exhibit significant changes in elastic modulus throughout the incu-
bation periods (p > 0.05). Following silver fluoride treatments, at post 
treatment time point, all treatment groups, except for the KI treatment 
group, increased the elastic modulus of demineralized dentin beams 
compared to their baseline measurements and the control group 
(p < 0.05). The elastic modulus for SDF and SF groups remained 
significantly higher than the control group across all incubation periods 
(p < 0.05). No significant differences in elastic modulus occurred be-
tween the incubation periods within the SDF and SF group (p > 0.05). 
The addition of KI significantly reduced the elastic modulus after 1-week 
incubation. SDF group produced a 3.2-fold increase in elastic modulus 
compared to SDF ‡ KI group, and SF group exhibited 2.7-fold increase in 
elastic modulus compared to SF ‡ KI group. SF ‡ KI group showed 
comparable elastic modulus with the control group up to 3 months in-
cubation (p > 0.05), however, at 6 months incubation, this group 
revealed significantly lower elastic modulus than the control group 
(p < 0.05). Both SDF ‡ KI and KI groups had lower elastic modulus re-
sults than the control starting from 1-week incubation (p < 0.05).
3.3. Total enzymatic activity
The results of the generic total MMP activity screening assay are 
shown in Fig. 3 as the percentage inactivation of total enzymatic ac-
tivity. Untreated demineralized dentin beams served as control group 
and revealed zero percent inactivation. All treatment groups demon-
strated significant reductions in total MMP activity compared to un-
treated control across all incubation periods (p < 0.05). Following silver 
fluoride treatments, the ammonia-based silver fluoride treatment groups 
(SDF and SDF ‡ KI) showed higher MMP inactivation than the water- 
based silver fluoride treatment groups (SF and SF ‡ KI) at post treat-
ment and 1 week incubation (p < 0.05). KI treatment alone revealed the 
lowest MMP inactivation at all incubation periods (p < 0.05). At 1 
month incubation, SF treatment showed lower MMP inactivation 
compared to SF ‡ KI, SDF, SDF ‡ KI groups (p < 0.05) and no statisti-
cally significant differences were found among the three groups 
(p > 0.05). After 3 months incubation, all treatment groups, except for 
the KI treatment group, showed significant inactivation of around 95 
 5 % (p < 0.05).
3.4. Solubilized of collagen telopeptides (CTX and ICTP)
Fig. 4 illustrated the release of the telopeptide CTX, produced by 
cathepsin-K activity in demineralized dentin beams among all groups 
and incubation periods. Untreated demineralized dentin beams served 
as control group. All treatment groups revealed higher CTX release 
compared to the control at all incubation periods (p < 0.05). Despite a 
decrease in CTX release over time for all treatment groups, the untreated 
control group still showed a cumulative CTX release 2.7-fold lower than 
SDF group, which exhibited the second lowest cumulative release 
(p < 0.05). After 1-week incubation, CC-mediated CTX release was 
significantly higher for the SDF ‡ KI, SF ‡ KI and KI groups (p < 0.05). 
After 1 month incubation, the CC-mediated CTX release remained higher 
Fig. 1. Cumulative loss of dry mass of demineralized dentin beams submitted to different silver fluoride treatments up to 6 months incubation. Untreated dem-
ineralized dentin beams served as control. The chart columns represent the mean values and the error bars represent standard deviations (n ˆ 10/group). Different 
capital letters indicate significant differences between treatments within incubation periods, according to Tukey test (p < 0.05). Different lowercase letters indicate 
significant differences between incubation times within treatment groups, according to Tukey test (p < 0.05). Abbreviations: SDF ˆ Silver Diamine Fluoride; KI 
ˆ Potassium Iodide; SF ˆ Aqueous Silver Fluoride.
M. Uctasli et al.                                                                                                                                                                                                                                 Dental Materials xxx (xxxx) xxx 
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for SDF ‡ KI and KI groups (p < 0.05), however, SF ‡ KI group revealed 
lower CTX release comparable with SF group (p > 0.05).
Release of the telopeptide ICTP by the endogenous MMPs for dem-
ineralized dentin beams are summarized in Fig. 5. Untreated deminer-
alized dentin beams served as control group and exhibited the lowest 
MMP-mediated ICTP release, with no significant differences noted 
compared to KI treatment group (p > 0.05). ICTP release decreased in 
the SDF and SF groups over time (p < 0.05); whereas MMP-mediated 
release increased progressively in SDF ‡ KI and SF‡KI treatment 
groups (p < 0.05). MMP-mediated ICTP release from SDF and SF groups 
up to 1-month incubation showed higher MMP-mediated degradation 
(p < 0.05). Groups combined with KI treatment revealed significantly 
higher MMP-mediated degradation at 3 months and 6 months incuba-
tion periods (p < 0.05).
3.5. Hydroxyproline quantification
Hydroxyproline (HYP) release for demineralized dentin beams 
among all groups and incubation periods is shown in Fig. 6. Untreated 
demineralized dentin beams served as control group. Analysis of HYP as 
an indicator of total collagen degradation revealed that SDF ‡ KI, SF 
‡ KI and KI groups and SDF and SF groups resulted in 10–20 fold and 2- 
fold increase in collagen degradation compared to the untreated control 
at 1 week incubation (p < 0.05), respectively. After 1 month of incu-
bation, SDF, SF and KI groups did not significantly affect collagen 
degradation, yielding results comparable to control group (p > 0.05). 
SDF ‡ KI and SF ‡ KI groups showed higher collagen degradation than 
SDF, SF and untreated control groups at all incubation periods 
(p < 0.05). At 1-month and 3-month incubations, SF ‡ KI resulted in 
Fig. 2. Elastic modulus means (MPa) and standard deviations of demineralized dentin beams submitted to different silver fluoride treatments up to 6 months in-
cubation. Untreated demineralized dentin beams served as control. The chart columns represent the mean values and the error bars represent standard deviations 
(n ˆ 10/group). Different capital letters indicate significant differences between treatments, within incubation periods, according to Tukey test (p < 0.05). Different 
lowercase letters indicate significant differences between incubation times within treatment groups, according to Tukey test (p < 0.05). Abbreviations: SDF ˆ Silver 
Diamine Fluoride; KI ˆ Potassium Iodide; SF ˆ Aqueous Silver Fluoride.
Fig. 3. Total enzymatic activity of dentin MMPs shown as the mean values of percentage inactivation of MMPs of all treatment groups up to 6 months of incubation. 
Untreated demineralized dentin beams served as control, 0 % MMP inactivation. The chart columns represent the mean values and the error bars represent standard 
deviations (n ˆ 10/group). Different capital letters indicate significant differences between treatments within incubation periods, according to Tukey test (p < 0.05). 
Different lowercase letters indicate significant differences between incubation times within treatment groups, according to Tukey test (p < 0.05). Abbreviations: SDF 
ˆ Silver Diamine Fluoride; KI ˆ Potassium Iodide; SF ˆ Aqueous Silver Fluoride.
M. Uctasli et al.                                                                                                                                                                                                                                 Dental Materials xxx (xxxx) xxx 
6 
significantly higher total collagen degradation than SDF ‡ KI group 
(p < 0.05).
3.6. Total extractable protein
Total extractable protein levels for demineralized dentin beams 
among all groups and incubation periods are shown in Fig. 7. Untreated 
demineralized dentin beams served as the control group. At 1-week in-
cubation, groups including the KI treatment revealed significantly 
higher protein levels than SDF, SF and untreated control group 
(p < 0.05). There were no significant differences in extractable protein 
levels between SDF group and untreated control group (p > 0.05), nor 
between SF group and untreated control group, both of which exhibited 
protein levels ranging from 2400 to 3000 ng protein/mg in dry dentin 
(p > 0.05). However, there was a significant difference between the SDF 
and SF groups that SF resulted in higher protein levels compared to SDF 
(p < 0.05). Protein loss decreased in all groups from 1 week to 2 months 
but then increased from 2 months to 3 months (p < 0.05). After 1 week 
Fig. 4. The rate of release of CTX fragment from demineralized dentin beams submitted to different silver fluoride treatments. Untreated demineralized dentin beams 
served as control. Aliquots of calcium- and zinc- containing media were analyzed after each incubation period. Values are pg telopeptide/mg dry dentin per unit time. 
The chart columns represent the mean values and the error bars represent standard deviations (n ˆ 10/group). Different capital letters indicate significant differences 
between treatments, within incubation periods, according to Tukey test (p < 0.05). Different lowercase letters indicate significant differences between incubation 
times within treatment groups, according to Tukey test (p < 0.05). Abbreviations: SDF ˆ Silver Diamine Fluoride; KI ˆ Potassium Iodide; SF ˆ Aqueous Sil-
ver Fluoride.
Fig. 5. The rate of release of ICTP fragment from demineralized dentin beams submitted to different silver fluoride treatments. Untreated demineralized dentin 
beams served as control. Aliquots of calcium- and zinc- containing media were analyzed after each incubation period. Values are pg telopeptide/mg dry dentin per 
unit time. The chart columns represent the mean values and the error bars represent standard deviations (n ˆ 10/group). Different capital letters indicate significant 
differences between treatments, within incubation periods, according to Tukey test (p < 0.05). Different lowercase letters indicate significant differences between 
incubation times within treatment groups, according to Tukey test (p < 0.05). Abbreviations: SDF ˆ Silver Diamine Fluoride; KI ˆ Potassium Iodide; SF ˆ Aqueous 
Silver Fluoride.
M. Uctasli et al.                                                                                                                                                                                                                                 Dental Materials xxx (xxxx) xxx 
7 
incubation, ammonia- and water-based silver fluoride treatments 
revealed lower protein loss compared to control group (p < 0.05). KI 
group revealed higher protein loss than corresponding control groups at 
all incubation periods (p < 0.05).
3.7. SEM analysis
Representative SEM micrographs for demineralized dentin surfaces 
following ammonia- and water-based silver fluoride treatments to 
evaluate the surface and the cross sectional view are shown in Fig. 8. 
H3PO4 etching revealed a thick layer of demineralized collagen for both 
untreated (control) and treated (SDF, SDF ‡ KI, SF, SF ‡ KI) groups 
(Fig. 8). Untreated demineralized dentin beams presented totally 
dissolved smear layer, producing clear tubule disobliteration without 
any precipitations (Fig. 8A, B). Silver precipitates were observed 
covering most of the surface and within the dentinal tubules after 
ammonia-based (SDF and SDF ‡ KI) and water-based (SF and SF ‡ KI) 
silver fluoride treatments (Fig. 8 C, D, E, F, G, H, I, J). Treatments 
including KI produced precipitates that were more densely packed and 
extended deeper in the dentinal tubules (Figure E, F, I, J).
3.8. Evaluation of gelatinase activity by in situ zymography
Representative confocal laser scanning microscopy images and the 
mean results of gelatinolytic activity expressed as the intensity of green 
fluorescence within hybrid layers (HL) are summarized in Fig. 9. The 
Fig. 6. Hydroxyproline (HYP) levels of demineralized dentin beams submitted to different silver fluoride treatments. Untreated demineralized dentin beams served 
as control. Aliquots of calcium- and zinc- containing media were analyzed after each incubation period. Values are ng HYP/mg dry dentin per unit time. The chart 
columns represent the mean values and the error bars represent standard deviations (n ˆ 10/group). Different capital letters indicate significant differences between 
treatments, within incubation periods, according to Tukey test (p < 0.05). Different lowercase letters indicate significant differences between incubation times within 
treatment groups, according to Tukey test (p < 0.05). Abbreviations: SDF ˆ Silver Diamine Fluoride; KI ˆ Potassium Iodide; SF ˆ Aqueous Silver Fluoride.
Fig. 7. Extractable protein levels from demineralized dentin beams submitted to different silver fluoride treatments. Untreated demineralized dentin beams served as 
control. Aliquots of calcium- and zinc- containing media were analyzed after each incubation period. Values are ng total protein/mg dry dentin per unit time. The 
chart columns represent the mean values and the error bars represent standard deviations (n ˆ 10/group). Different capital letters indicate significant differences 
between treatments, within incubation periods, according to Tukey test (p < 0.05). Different lowercase letters indicate significant differences between incubation 
times within treatment groups, according to Tukey test (p < 0.05). Abbreviations: SDF ˆ Silver Diamine Fluoride; KI ˆ Potassium Iodide; SF ˆ Aqueous Sil-
ver Fluoride.
M. Uctasli et al.                                                                                                                                                                                                                                 Dental Materials xxx (xxxx) xxx 
8 
untreated 5 s H3PO4 -etched group served as control. In all groups, green 
fluorescence within the HL as well as underlying dentinal tubules were 
detected. In situ zymography images using confocal microscopy revealed 
a decrease in gelatinase activity at ammonia- and water-based silver 
fluoride treated groups compared to the untreated control (p < 0.05). KI 
treatment increased the gelatinolytic activity compared to SDF and SF 
(p < 0.05). SF ‡ KI treatment revealed the highest gelatinolytic activity 
among treatment groups (p < 0.05).
4. Discussion
This study focused on the efficacy of ammonia- and water-based 
silver fluoride treatments in preventing dentin matrix degradation. 
The null hypothesis was rejected, since ammonia- and water-based silver 
fluoride treatments affected the activity of dentinal MMPs and CCs, the 
loss of dry mass, elastic modulus, the degradation of protein and 
collagen structure in demineralized dentin matrix.
The measurement of dry dentin mass loss and the modulus of elas-
ticity using three-point bending test was selected to evaluate the 
degradation of demineralized dentin matrices and the mechanical sta-
bility of collagen [32], respectively. A decrease in dry dentin mass and 
modulus of elasticity indicates compromised mechanical properties, 
primarily due to changes in the collagen structure. These methods allow 
repeated measurements on the same specimen over time, providing 
insight into the long-term effects of different treatments [20]. In the 
current study, dry mass loss and elastic modulus revealed similar trends, 
silver fluoride formulations and the time elapsed after treatments were 
critical factors that influence the structural integrity of demineralized 
dentin. Immediate after treatments, an increase in the elastic modulus of 
all treated groups were observed, correlating with the deposition of 
silver particles within dentin tubules (Fig. 8). Among the tested silver 
fluoride treatments, ammonia-based SDF and water-based SF demon-
strated stable dry mass loss and elastic modulus values over the studied 
period. However, the addition of KI treatment as the second step exac-
erbated dry mass loss and elastic modulus of the dentin organic matrix. 
This effect could be explained with denser material deposition on the 
dentin surface, which extended inside the dentinal tubules (Fig. 8 F; J) 
and possibly interfere with the collagen matrix. Based on previous 
studies, there is no consensus regarding ammonia- and water-based 
silver fluoride treatments depth of penetration into dentin tubules 
[33]. On the groups included the KI application, visual observations 
during the experiment revealed increased transparency and signs of 
structural degradation, such as cracks along the dentin surface, after 
1-month incubation period. SF ‡ KI treatment showed better results 
than the SDF ‡ KI treatment, this could be explained by the differences 
in water and ammonia content between the solutions, respectively. 
Clinically, water-based silver fluoride treatments offer benefits such as 
reduced odor, less irritation and neutral pH, which can enhance both 
clinician and patient comfort.
A generic MMP Assay kit was used for the detection of total matrix 
bound MMP activity enables rapid screening of enzymatic activity using 
a colorimetric assay and allows large number of specimens to be eval-
uated at the same time. This technique detects the total activity of a 
variety of MMPs and provides direct information about the efficiency of 
MMP activation or inactivation [34]. The assay can detect total MMP 
activity; however, it does not differentiate the specific MMP isoforms. In 
the current experiment, demineralized dentin beams were used as the 
MMP source instead of recombinant human MMPs [13], which enhances 
the clinical relevance of the study. All silver fluoride treatments were 
effective in inactivating MMP activity, which aligns with the short-term 
findings of other studies [14]. Consistent with the findings reported by 
Mei et al. [13] MMP inhibition was observed with 38 % (w/v) silver 
fluoride concentration [13].
To date, no study has evaluated MMP activity using an MMP assay kit 
for water-based silver fluoride. In short term, we found that ammonia- 
based silver fluoride was more effective, which is in agreement with 
Fig. 8. Representative SEM micrographs showing the surface and cross- 
sectional views of ammonia- (SDF and SDF ‡ KI) and water- (SF and SF ‡
KI) based silver fluoride treatments. Abbreviations: SDF ˆ Silver Diamine 
Fluoride; KI ˆ Potassium Iodide; SF ˆ Aqueous Silver Fluoride.
M. Uctasli et al.                                                                                                                                                                                                                                 Dental Materials xxx (xxxx) xxx 
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the in situ zymography results (Fig. 9) and is consistent with findings 
from another study [16]. The lower effectiveness of water-based silver 
fluoride (SF and SF ‡ KI) in inactivating MMP activity compared to 
ammonia-based silver fluoride (SDF and SDF ‡ KI) could be explained 
by the higher water content of the water-based formulation [31]. In 
long-term incubation, our findings confirm that both ammonia- and 
water-based silver fluoride treatments decreased the total enzymatic 
activity. Unlike other crosslinking methods which require relatively long 
application times and therefore clinically impractical [35], silver fluo-
ride treatments are viable alternatives as they can be applied to dentin 
within minutes while remaining effective in inactivating total enzymatic 
activity [36,37].
To evaluate collagen degradation, a radioimmunoassay (RIA) and an 
enzyme-linked immunosorbent assay (ELISAs) were used to quantify 
enzyme-specific degradation products of collagen, specifically C-termi-
nal telopeptide fragments in the incubation media. Both cysteine ca-
thepsins (CCs) and matrix metalloproteinases (MMPs) can cleave type I 
collagen at distinct sites. MMPs generate ICTP (C-terminal cross-linked 
telopeptide of type I collagen), whereas cathepsin K produces CTX (C- 
terminal telopeptide of type I collagen) [3,26,32]. These markers are 
widely utilized to identify the enzymatic pathways involved in collagen 
degradation. Notably, KI-treated demineralized dentin beams exhibited 
an even greater increase in cathepsin K-mediated CTX release during 
short-term incubation. The KI-treated demineralized dentin beams 
exhibited lower levels of MMP-mediated ICTP release. An inverse rela-
tionship between ICTP and CTX release trends was observed. However, 
the exact mechanism behind this fluctuation remains unclear, as dentin 
protease activity is influenced not only by MMPs and CCs [32]. 
Acid-etching procedures and acidic monomers in dental adhesive tech-
niques expose endogenous dentin proteases, facilitating their activation 
upon contact with silver fluoride. The commercially available 
ammonia-based silver fluoride is an alkaline solution (Table 1), which 
could influence the MMP activity [13]. Since MMPs function optimally 
at neutral pH, the alkaline pH of ammonia-based silver fluoride may 
alter their activity. MMPs, a family of zinc- and calcium-dependent en-
dopeptidases present in dentin and saliva, are capable of degrading both 
native and denatured collagen [19,20], therefore, zinc- and 
calcium-containing artificial saliva were used as the incubation medium 
in the current study. Designated incubation periods of 1 week, 1 month, 
3 months and 6 months were selected to represent early, intermediate 
and long-term effect of dentin matrix degradation after silver-fluoride 
based treatments [18,21,38].
Analysis of hydroxyproline (HYP) as an indicator of total collagen 
degradation revealed that the amount of released collagen was higher in 
all groups compared to demineralized dentin during the short-term in-
cubation period. Notably, KI-treated groups exhibited higher collagen 
release, indicating that KI has a definite effect on collagen degradation. 
The hydroxyproline assay further revealed that groups treated with 
ammonia- and water-based silver fluoride combined with KI consistently 
showed greater total collagen degradation than groups that did not 
contain KI, a chemical reaction between KI and silver fluoride treat-
ments may be responsible for this effect. Furthermore, the SF ‡ KI group 
exhibited lower collagen degradation than the SDF ‡ KI group. This 
trend suggests that the interaction between ammonia and water-based 
silver fluoride formulations influence collagen degradation.
The Bradford protein assay is one of the most used methods for 
measuring protein content, first introduced by Bradford in 1976 and has 
been widely applied to quantify protein concentrations [29]. Bradford 
assay results showed similar trends observed in elastic modulus and dry 
mass measurements. KI exhibited excessive protein release, suggesting 
Fig. 9. A. Representative in situ zymography images acquired at green channel (A;B;C;D;E), differential interference contrast (DIC) (F;G;H;I;J) and merged channels 
(K;L;M;N;O) of ammonia- (SDF and SDF ‡ KI) and water- (SF and SF ‡ KI) based silver fluoride treatments. B. Gelatinolytic activity expressed as intensity of green 
fluorescence (pixels/µm2) within the hybrid layer. Chart columns represent the mean values and the error bars represent standard deviations of gelatinolytic activity. 
The untreated group served as control. Different capital letters indicate significant differences between treatments according to Tukey test (p < 0.05). Abbreviations: 
SDF ˆ Silver Diamine Fluoride; KI ˆ Potassium Iodide; SF ˆ Aqueous Silver Fluoride; R ˆ Resin composite; HL ˆ Hybrid layer; D ˆ Dentin.
M. Uctasli et al.                                                                                                                                                                                                                                 Dental Materials xxx (xxxx) xxx 
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that KI contributes to short-term protein degradation before stabilizing 
over time.
The use of KI as the second step after ammonia- and water-based 
silver fluoride were found to negatively impact dentin integrity. Spe-
cifically, the addition of KI was associated with increased dry mass loss, 
decreased apparent modulus of elasticity and elevated CC-mediated CTX 
and MMP-mediated ICTP degradation. Moreover, hydroxyproline and 
total protein levels indicated that KI treatment accelerated collagen 
matrix degradation. It has been reported that KI reduces the effective-
ness of SDF in arresting or preventing caries [39]. Therefore, the clinical 
use of KI should be carefully reconsidered, particularly in cases where 
priority is not esthetics [40]. Moreover, silver fluoride is commonly used 
in high-caries-risk patients and has demonstrated efficacy in preventing 
and arresting both early childhood and root caries in the elderly [41,42]. 
In such cases, esthetic concerns are often secondary [40]. 
Ammonia-based silver diamine fluoride (SDF) did not cause an imme-
diate color change when applied to demineralized dentin, whereas 
water-based silver fluoride (SF) resulted in a noticeable yellowish 
discoloration upon contact with collagen. This discoloration is likely 
attributed to a chemical interaction between silver ions and exposed 
collagen, potentially affecting the bonding interface and long-term sta-
bility of the material [14,16].
Although KI treatment alone has no direct clinical application, it was 
included as a separate group to assess its effect on demineralized dentin 
and to determine whether KI alone contributes to collagen matrix 
degradation. Our findings revealed that KI treatment alone had a 
measurable impact on dentin integrity. Specifically, KI significantly 
increased dry mass loss and total extractable protein levels, suggesting 
that KI may contribute to dentin dissolution and interfere with protein 
release. The observed weight loss reflects alterations in both organic and 
inorganic components of dentin. As an ionic agent, KI interacts with 
collagen and KI role in modifying dentin composition requires further 
investigation. Regarding elastic modulus, total enzymatic activity and 
MMP-mediated ICTP release, KI treatment alone did not appear to have 
a direct effect. However, KI influenced CTX degradation for up to 6 
months and significantly increased CC-mediated CTX release and hy-
droxyproline release after 1 week of incubation. To our knowledge, no 
study has evaluated the effect of KI treatment alone on dentin collagen 
matrix. KI, commonly applied to address tooth’s discoloration issues, 
was not evaluated alone on dentin using scanning electron microscopy 
or in situ zymography. Therefore, future studies should investigate KI 
treatment independently regarding collagen integrity. Moreover, novel 
silver-based materials should be developed to address discoloration 
while preserving the therapeutic benefits of silver fluoride treatments 
[43,44].
Silver fluoride treatments were initially introduced to the market for 
the management of dentin hypersensitivity, that explain the precipita-
tion of silver on the dentin surface and within dentinal tubules (Fig. 8). 
Notably, the SEM specimens were rinsed following silver fluoride 
treatments, mimicking the clinical scenario where no restorative mate-
rial is subsequently placed. Furthermore, the high density of silver 
particles observed within the dentinal tubules in the cross-sectional view 
can be attributed to the use of fully demineralized dentin beams.
Gelatinolytic activity in hybrid layers using in situ zymography was 
first described by Mazzoni et al. [31]. In situ zymography images using 
confocal microscopy revealed a decrease in gelatinase activity in silver 
fluoride treatment groups compared to the untreated control group. 
Moreover, enzymatic activity was higher in water-based silver fluoride 
treatment. These findings are in agreement with total endogenous 
enzymatic activity. The alkaline pH of ammonia-based and the neutral 
pH of water-based silver fluoride could interfere with the collagen ma-
trix (Table 1). Hence, it is reasonable to assume that lower pH may 
modify silver-crystals formation on demineralized dentin when 
compared to higher pH [14]. It can be speculated that the high alkalinity 
of SDF contributes to the neutralization of the acidity of demineralized 
dentin, which may reduce endogenous proteinase-related collagen 
degradation [4,14]. In the present study, only immediate collagenolytic 
activity was evaluated. However, assessing the enzymatic activity using 
in situ zymography over a longer period would be beneficial, as Ales-
sandro et al. [16] conducted a one-year study and reported comparable 
findings. Ideally, using the same tooth for all groups would improve 
consistency. To minimize this limitation, non-fluorescent gels were 
analyzed to subtract each tooth’s intrinsic gelatinolytic activity, thereby 
enabling more accurate comparison across different teeth.
A material’s ability to reduce the degradation of dentin’s organic 
matrix can be indirectly associated with the longevity of composite 
fillings [45]. Currently, there is no universally accepted clinical protocol 
for the application of ammonia- and water-based silver fluoride treat-
ments. Depending on the clinical scenario, ammonia- and water-based 
silver fluoride treatments may either be left without subsequent 
restorative procedures or be followed by restoration with glass ionomer 
cement or composite fillings [46]. Considering the compelling evidence 
that silver fluoride treatments may offer a protective effect on collagen 
structure, future in vitro and in vivo studies should focus on evaluating 
long-term resin bonding outcomes to allow clinicians to balance the 
potential benefits silver fluoride treatments on dentin integrity and 
bonding performance. Understanding the mechanisms of silver 
fluoride-dentin interactions regarding different concentrations, formu-
lations and application protocols remain necessary before future clinical 
applications.
5. Conclusion
The inhibitory efficacy of ammonia- and water-based silver fluoride 
treatments on dentin protease activity should not be overestimated. 
Both ammonia-based and water-based silver fluoride treatments, 
particularly when combined with potassium iodide, revealed complex 
effects on dentin protease activity and collagen matrix integrity. Silver 
fluoride treatments do not necessarily fully inhibit endogenous protease 
degradation of demineralized collagen; however, their use can provide 
long-term protection regarding collagen’s mechanical properties and 
overall structural integrity. The use of silver-based cariostatic agents 
associated to potassium iodine in efforts to prevent collagen degradation 
should be avoided.
Declaration of Competing Interest
The authors declare that they have no known competing financial 
interests or personal relationships that could have appeared to influence 
the work reported in this paper.
Acknowledgements
This work was supported by EVO-funding of Turku University Hos-
pital, Finland (#13140).
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