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\begin{document}

\title{Influence of Matrix Metalloproteinases content on eardrum atrophy in
Otitis Media with Effusion in children}



\author[1]{Lylou Casteil}%
\author[2]{Corentin Affortit}%
\author[1]{Jean Luc Puel}%
\author[1]{Fabian Blanc}%
\author[1]{Frederic Venail}%
\author[1]{Jing Wang}%
\author[1]{Michel Mondain}%
\affil[1]{Université de Montpellier}%
\affil[2]{Institut des Neurosciences de Montpellier}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Objectives Otitis media with effusion (OME) is a common pathology in
children. Effusions contain Metalloproteinases (MMPs), which can lead to
atrophy of the tympanic membrane (TM) due to destructive effect on the
lamina propria. Not all cases of OME are complicated with atrophy, maybe
explained by an inter-individual variation of MMP concentration in
effusions. The purpose of this study was to determine the correlation
between concentration of MMPs and existence of TM atrophy in children.
Participants - Main outcome measures The effusion from thirty middle
ears were collected during insertion of VT in children aged 15 months to
10 years, including 11 eardrums with tympanic atrophy and 19 without
tympanic atrophy. ELISA tests were used to measure concentrations of
MMP-2, MMP-7, MMP-9 and TIMP-2 in the effusions. Correlations between
MMP levels and atrophy of eardrum was investigated, as well as
correlation with age, gender, number of previous ventilation tube
insertion, and viscosity of the glue. Results The mean concentration of
MMP-2 was higher in the atrophic group than in effusions without TM
atrophy (0.6 ng/mg versus 0.5 ng/mg total protein respectively), while
the TIMP-2 concentration was lower in this group. The level of MMP-2
decreased with the age. Finally, a significantly higher concentration of
MMP-9 and TIMP-2 was found in high-viscosity effusions. Conclusion This
study suggests that MMP-2 activity could play an important role in
destruction of the eardrum during OME in infancy. MMP-2 level assessment
could be interesting for determining children with risk of lamina
propria destruction.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Otitis media with effusion (OME) is a chronic otitis media with
inflammatory effusion in the middle ear. Inflammation causes a
thickening of the mucosa within the middle ear cleft which, by modifying
the gas exchange capacities, is responsible for a decrease in the
partial pressure of oxygen facilitating the retraction of the tympanic
membrane (TM) (1). In children, inflammation often arises via an
infectious origin by contamination of middle ear cavities via the
Eustachian tube.

The diagnosis of OME is clinical, by otoscopy and by tympanometry (2).
The exact incidence of OME is unknown, due to the nature of the disease
which can be asymptomatic. However, it is thought to be up to 50\%
children under 12 months of age and 60\% children at the age of 2 years.
A study conducted with systematic screening of children aged 1 to 4
years suggests a prevalence between 15 and 40\% (3).

OME can lead to structural changes in the TM (4)(17). Tympanosclerosis
plaques are classic and not dangerous, but atrophy of the eardrum can
lead to retraction pockets, and then into more significant disease such
as cholesteatoma or chronic~adhesive otitis media (5) (6). In otoscopy,
atrophy appears as a thin, partially or totally transparent eardrum (8).
Histologically, the atrophy of the pars tensa is explained by the
destruction of type IV collagen fibers of the lamina propria (7), and it
has been suggested that the activity of matrix metalloproteinase are
involved in destruction of tympanic membrane fibrous layer (9).

An increase in matrix metalloproteinase (MMP) activity may be present in
OME. MMPs are a multigenic family (25 members and 7 different classes)
of zinc-dependent, secretory or membrane enzymes. They control the
activity of extracellular matrix proteins by proteolytic cleavage. Their
targets include membrane-bound or soluble molecules involved in the
transmission of intercellular signals such as cytokines, chemokines,
trophic factors, adherence proteins and different receptors. Correlation
has been demonstrated between significant activity of MMP-2 and 9
(gelatinases), MMP-3 (stromelysin 1) and 7 (matrilysin 1) and MMP-8
(collagenase-2) and the viscosity of the effusion (7) (10) (11) (12).
Each MMP is specific to one or more component of the extracellular
matrix (13). Gelatinases (MMP-2, MMP-9) are the fourth class of MMPs,
and their proteolytic activity is directed against denatured
interstitial collagen (gelatin) and type IV and V collagens of basal
membranes. MMP-9 (gelatinase-B) expression is low or absent in normal
tissues and limited to monocytes and macrophages. MMP-7 (matrilysin-1)
is part of the matrilysin group, and is specifically expressed by
tumoral epithelial cells. Its proteolytic spectra are partially
divergent but include fibronectin and gelatin.

In tissues, the proteolytic activity of MMPs is controlled by four
inhibitors called TIMPs (tissue inhibitors of metalloproteinases) (14).
TIMPs (TIMP-1 to TIMP-4) are endogenous inhibitors of activated MMPs.
The MMP/TIMP system controls the cell-cell and cell-matrix interactions
involved in many physiological processes, including proliferation,
differentiation, migration and cell death. TIMP-2 works as an MMP
inhibitor but also as an activator since it inhibits the active form of
MMPs and activates proMMPs. Thanks to their C-terminal domain, there is
a specificity between MMP and TIMP types, TIMP-2 is an inhibitor that
binds specifically to MMP-2.

In France, insertion of VT is the second most frequent ENT surgery
procedure after tonsillectomies and/or adenoidectomies. This surgery is
indicated for auditory and/or infectious reasons and/or OME complicated
with TM atrophy with posterior mesotympanic retraction (2). Studies are
inconclusive about the effectiveness of VTs on the development of
tympanic atrophy (15) (16). The inter-individual variability of the
lytic activity of OME liquids, both in its composition and/or enzymatic
concentration, could explain the difficulty to statistically demonstrate
the efficacy of VT in preventing the development of atrophy.

The purpose of this study was to assess a correlation between the
presence of eardrum atrophy and the level of MMP-2, MMP-9, MMP-7, TIMP-2
in OME effusion. The secondary objective was to determine if there is a
relationship between the level of MMPs and TIMP-2 and the patient's age,
gender, history of VT insertion, thickness of the glue (mucous or
serous), and hearing loss.

\textbf{METHODS}

This is a pilot study and it is prospective, monocentric, observational
and case-controlled.

\textbf{Population}

Thirty-five children, 12 girls and 23 boys, whose indications for VT
were selected according to SFORL recommendations (French ENT
Society)(18), were included in this study (Figure 1).

Five children were excluded (4 for insufficient volume to allow
analysis, 1 for blood contamination of the effusion). Of the 30
remaining children, a collection of 30 OME fluid samples was collected,
11 from ears with tympanic atrophy (preoperative examination under
microscope done by two different examiners) and 19 from ears without
tympanic atrophy (preoperative examination performed by two different
examiners finding thinness from visual aspect under microscope).

For each patient, the effusion was collected from both ears prior to VT
insertion but only one ear was analysed per child. In case of unilateral
TM atrophy, only this sample was analysed. In the event of bilateral
atrophy or healthy bilateral eardrum, the laterality of the effusion to
be analysed was selected by a coin toss.

The study population ranged from 15 months to 10 years of age. Eighteen
right ears and 12 left ears were selected. There were no differences
(Chi-2 test with Prism's Biostatgv and Graphpad software) between the
two groups (healthy and atrophic eardrums) in gender (p=0.28), age
(p=0.881), audiometric loss (p=0.299), or number of VT insertions in the
history (p=0.62 with the chi2 test and with a Cramer V=0.01). The
effusion was more mucous than serous in both groups (serous in 18\% of
cases in atrophic patients and 15\% in healthy patients p=0.865).

\textbf{Liquid collection protocol}

The procedure was performed under general anaesthesia. After cleaning
and washing the ear canal with saline solution, each effusion was
collected by aspiration with a 1.2 gauge suction cannula connected to a
cell trap, following myringotomy. The glue was considered mucosal if it
was thick, difficult to aspirate and stuck to the wall of the cell trap
(19) (requiring micro forceps to collect the effusion if necessary). It
was considered serous if the suction was easy and the collection was
liquid and mobile in the cell trap.

The samples collected were immediately immersed in a container filled
with ice to prevent protein denaturation by heat, and then transported
immediately to the laboratory. Each mucoid effusion was separated into
two samples, using micro scissors, and then placed in two cryotubes
marked with the patient's anonymised code. The thinner serous samples
were divided into two cryotubes by pipette. No dilution was carried out
at this stage. The samples had an average volume of 200\selectlanguage{greek}µ\selectlanguage{english}l. The cryotubes
were frozen at -80\selectlanguage{ngerman}°C.

\textbf{Dosing protocol - ELISA technique}

An ELISA kit was used (R\&D system Quantikine ELISA) for a quantitative
analysis of the total MMP-2, MMP-7 and MMP-9 (free and complex forms)
and human TIMP-2 (plate of 96 wells pre-coated with the primary
antibody). Conditioning was performed using a saline / EDTA buffer in
order to obtain viscosity of the samples to be compatible with this
technique. Samples were diluted in a buffer with 0.1 M NaCl, 1mM PMSF,
1x LAP, 5mM EDTA, 20mM Tris 0.6M. Thorough homogenization of the
effusions was achieved using a pestle, to manually extract the proteins.
The samples were then placed on a circular stirrer for 1 hour at 4°C. A
total protein assay was performed using BCA kits (BCA protein assay kit,
ThermoFisher Scientific®) on each buffered sample.

We used 1/10 and 1/20 dilutions of the middle ear effusions for the
analysis of MMP-2, TIMP-2 and MMP-7 concentrations, and 1/40 for the
determination of MMP-9. On each plate, 64 ELISA wells were used to test
the duplicate samples. The remaining 16 wells were used to carry out the
standard curve with standard solutions.

Analysis was performed by spectrophotometry at 450 nm and 540 nm. The
optical density (OD) was the average of the two ODs obtained by each
duplicated sample on the microplate reader. A concentration in ng/mL was
obtained (ratio of OD to standard curve) ), then this level was
normalized by the total protein level of the sample, the final
concentration being expressed in ng/\selectlanguage{greek}µ\selectlanguage{english}g protein.

Results are presented as an average concentration and standard
deviation. The statistical data were calculated using a non parametric
test (Mann-Whitney test for independent data).

\textbf{Ethical and regulatory aspects}

This project has been validated by the Local Ethics Committee (IRB of
Montpellier) in MR-004. An explanatory note was given to the parents and
children included in the study, and consent was collected at the end of
the consultation.

\textbf{RESULTS}

\textbf{Determination of MMP-2, MMP-7, MMP-9 and TIMPS 2 levels (Table
1\&2)}

The average MMP-2 level was lower in the effusions from non-atrophic TMs
(average 85,4 ng/ml) than in the effusions from atrophic TMs (113,2
ng/ml), whether the concentration is expressed in ml of effusion or
related to the total protein content: this difference was not
significative between the two groups (p=0.28).

The mean concentration of TIMP-2 was higher in healthy subjects (mean
73,7 ng/ml -- 0,64 ng/\selectlanguage{greek}µ\selectlanguage{english}g total protein) than in the atrophic TMs group
(65,8 ng/ml -- 0,51 ng/\selectlanguage{greek}µ\selectlanguage{english}g total protein). There is no significant
difference between the two groups of samples (p=0.76).

The average MMP-7 level was 71 ng/ml in the non-atrophic samples (0,59
ng/\selectlanguage{greek}µ\selectlanguage{english}g total protein) vs. 68,5 ng/ml in the atrophic group (0,48 ng/\selectlanguage{greek}µ\selectlanguage{english}g
total protein): the difference is not significant between the two groups
(p=0.68).

The quantities of MMP-9 measured in the samples were higher than for
MMP-2 and MMP-7. The average MMP-9 level was 1120 ng/ml in the
non-atrophic group (110 ng/\selectlanguage{greek}µ\selectlanguage{english}g total protein) vs. 822,6 ng/ml in the
atrophic TMs samples (0,48 ng/\selectlanguage{greek}µ\selectlanguage{english}g total protein). However, this
difference was not significant (p=0.73).

\subsection*{}

{\label{section}}

\textbf{Clinical predictive factors for MMPs and TIMP-2 concentration}

MMP-2 and TIMP-2 level are negatively correlated to the age of the child
(Figure 2). However, this difference was not significative (p= 0,08) and
the ratio MMP-2 / TIMP-2 remains constant among ages. MMP and TIMP-2
rates were not influenced by the gender, or the preoperative conductive
hearing loss.

The MMP-7 rate was significantly higher in children who no history of
previous VT insertion (p = 0.013). This trend is not significant for
MMP-2 and TIMP-2 (p=0.19).

The MMP and TIMP-2 levels were higher in the thicker, more mucous,
effusions regardless of the condition of the eardrum. This difference is
significant for MMP-9 and TIMP-2 (p=0.0017 and p=0.013 respectively).

\section*{DISCUSSION}

{\label{discussion}}

OME is a major public health problem in paediatric ENT practice (8) and
understanding the mechanism of tympanic complications is fundamental in
reducing the sequelae of this disease (5). Proteolytic enzymes can play
an important role in the development of TM atrophy associated with OME,
and then into cholesteatoma. Many authors refer to the role of proteases
in the pathogenesis of OME complications (9--11,15,20). Our study
suggests a non-significant link between the level of MMP-2 in OME fluid
and the existence of tympanic atrophy. A higher concentration of MMP-2
in the effusions of patients with atrophic tympanic membranes has been
found. Its specific inhibitor TIMP-2, assessed in order to estimate the
active MMP-2 level without the inactive pro-enzymes (14), was also lower
in the effusions of patients with atrophic tympanic membranes. The MMP-9
level tended to be lower in children with atrophy, while the MMP-7 rate
was quite similar in both groups. So, the MMP-2 enzyme activity tended
to be higher in cases of tympanic atrophy, even if the difference was
not significant.

The ELISA technique was used for the assays. Gelatin or casein
zymography is an electrophoresis examination frequently performed in
other studies to measure MMP activity, because it dissociates MMPs from
TIMPs. However, ELISA, which is also commonly used for this type of
assay, is a validated technique. The MMP concentrations measured in this
study in ng/\selectlanguage{greek}µ\selectlanguage{english}l were consistent with MMP levels found in previous
publications, except for the MMP-9 concentrations which were higher than
in other studies. A South Korean study (21) studying MMP-9 levels in OME
with and without allergy found also high concentrations ranging from 100
to 920 pg/mg total protein.

Destruction of fibrous layer in OME may be related to the duration of
the OME's evolution (5), but this cannot be demonstrated in the general
population because OME is often asymptomatic. In the literature, the
duration of OME is often estimated from the number of VT insertions, and
the age of the patients. In our study, MMP2 and TIMP-2 concentrations
tended to decrease with age (figure 2), as reported in a 1994 American
study (16).However, the ration MMP-2/TIMP-2 did not vary among age.

In our study, the number of previous VT was not correlated with the MMP
level, but the MMP-7 rate which was significantly higher in children who
had no prior history of VT placement. The study by Jennings and al. (20)
found an increase in MMP-2 levels with the number of VT operations,
while a Swedish study by Juhn and al. (16) found no relationship between
the number of VT insertions and MMP-2 and 9 concentrations.

In our study, a significantly higher concentration of MMP was found in
the thicker mucous effusions, the difference being significative for
MMP-9 and TIMP-2 (p=0.0017 -- p=0,013 respectively). This result has
been demonstrated in other studies (9,10,15,20). Interleukins such as
TNF\selectlanguage{greek}α \selectlanguage{english}and IL-1 are present in higher concentrations in mucous effusions
(16). The role of these molecules in inducing the transcription of MMPs
is consistent with the results of our research. On the other hand, our
results for TIMP-2 concentrations are at odds with the results from
Moon's work, since we found a significant link between elevated TIMP-2
concentrations and mucous effusions (9).

One of the limitations of our study is assessment of the degree of
tympanic atrophy by examination under a microscope, even with an
examination under microscope performed by two different examiners. The
gold standard would be to perform biopsies of the eardrum (4). However,
this could lead to perforations and is questionable in children. In
further studies it could be possible to analyse an animal model to
quantify the relationship between the degree of eardrum atrophy and MMP
concentrations in OME effusion.

The second limitation was the number of children enrolled: inclusion of
more subjects would increase the strength of our study. In addition, it
would be interesting to compare intra-individual MMP-2 levels in
children with unilateral tympanic atrophy, the child being his own
control.

\section*{CONCLUSIONS}

{\label{conclusions}}

This study showed a higher MMP-2 concentration in children presenting
with tympanic atrophy, while the TIMP-2 concentration was lower in this
group, suggesting a role of MMP-2 in the digestion of type IV collagen
from the basement membrane of the tympanum. This study also suggests a
higher concentration of MMP-9 and TIMP-2 in case of thicker, mucous
effusions. The level of MMP-2 decreased with the age, suggesting more
aggressive OME in young children leading to atrophy. In the future,
MMP-2 concentration assessment could refine the indications for the
insertion of VT in children to prevent the emergence of cholesteatoma.

\par\null

\section*{REFERENCES}

{\label{references}}

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