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Evaluation of Esterified Hyaluronic Acid as Middle Ear–Packing Material
Geming Li, MD;
Joseph G. Feghali, MD;
Elizabeth Dinces, MD;
John McElveen, MD;
Thomas R. Van De Water, PhD
Arch Otolaryngol Head Neck Surg. 2001;127:534-539.
ABSTRACT
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Objective To evaluate the efficacy of esterified hyaluronic acid (MeroGel) as
a middle ear (ME)–packing material.
Design Randomized controlled trial.
Material Twenty-four guinea pigs.
Intervention Group 1, MeroGel-treated animals (n = 10), bilateral wounding of ME
mucosa with 5 of the animals receiving the MeroGel packing in the left ME
and 5 of the animals receiving MeroGel in the right ME; group 2, absorbable
gelatin sponge–treated animals (n = 10), with the same experimental
protocol as in group 1 except that the absorbable gelatin sponge was the packing
material; group 3, untreated animals (n = 4), unilateral wounding of the left
ME mucosa in 2 animals and in 2 animals in the right ME, with no packing material.
Auditory brainstem recordings were performed for all groups before the ME
operation and 5 days and 6 weeks after the operation.
Results Auditory brainstem response recordings at postoperative day 5 showed
that all ears with ME packing had hearing losses in the frequency range of
500 to 4000 Hz. The recovery of hearing acuity at postoperative week 6 was
significantly better in group 1 (MeroGel-treated) guinea pigs compared with
group 2 (the absorbable gelatin sponge–treated) animals. In group 2
animals, 20% of the packing material remained in the ME cavities and new bone
formation was observed, while in group 1 animals, there was less packing material
in the ME and no formation of new bone.
Conclusions MeroGel is a nonototoxic packing material with a high level of biocompatibility
for ME mucosa; it is an effective supportive material following ME surgery
and is easily expelled from the ME cavity.
INTRODUCTION
HYALURONIC ACID (HA) is a naturally occurring extracellular polysaccharide
with a molecular weight in the range of 2 to 4 million daltons. Hyaluronic
acid is a component of the extracellular matrix of many tissues within the
body,1 making it highly biocompatible. Hyaluronic
acid is frequently used as a supportive aid in ophthalmologic surgery.2 MeroGel (Medtronic Xomed Inc, Jacksonville, Fla) is
made from an esterified form of HA (HYAFF; Fidia Advanced Biopolymer srl,
Abano Terme, Italy).
The supportive material commonly used since the late 1950s in otological
surgery is the absorbable gelatin sponge (AGS) that is manufactured from a
selected grade of animal skin gelatin. There are, however, some reports that
indicate possible deleterious effects secondary to the use of AGS in the middle
ears (MEs) of animals3 and in patients following
a stapedectomy.4, 5
There is an interest in using HA as a supportive material in ME surgery.
It is highly biocompatible and seems to have no ototoxic effect on the inner
ear.6, 7, 8 Hyaluronic
acid has also been used as packing in clinical otological surgery and in animal
experiments.9, 10 These studies
have suggested that HA was equivalent to or superior to AGS.
The aims of this study were 3-fold to: evaluate MeroGel as a supportive
substance during wound healing in the ME, determine if MeroGel has any toxic
effects on either ME mucosa or the hearing threshold, and compare the effects
of MeroGel packing with those of AGS packing on wound healing under identical
laboratory conditions.
MATERIAL AND METHODS
ANIMALS
All animals in this study were treated in accord with the guidelines
of the Albert Einstein College of Medicine, Institutional Animal Care and
Use Committee, Bronx, NY. Twenty-four, 4-week-old guinea pigs (Dunkin Hartley,
Charles River, Wilmington, Mass), weighing from 300 to 350 g, were divided
into 3 groups. Group 1 (MeroGel recipients) consisted of 10 guinea pigs that
underwent bilateral wounding of ME mucosa. Five animals received MeroGel packing
in their left ME and the remaining 5 animals received MeroGel packing in their
right ear. Group 2 (AGS packing recipients; Pharmacia & Upjohn Inc, Stockholm,
Sweden) consisted of 10 guinea pigs, with the same experimental plan as in
group 1 except AGS was used to pack the ME cavities. The volume and positioning
of packing material used in the MEs was standardized for the animals in groups
1 and 2. Group 3 (untreated) consisted of 4 guinea pigs with unilateral wounding
of the ME mucosa. In 2 guinea pigs the left ear was wounded and in the remaining
2 animals the right ear was wounded. No packing material was placed in the
MEs of group 3 animals.
AUDITORY BRAINSTEM RESPONSE RECORDINGS (ABR)
A series of 3 ABR tests for pure-tone stimuli were performed just prior
to the ME wounding operations, on postoperative day 5 and at postoperative
week 6 week. For each ABR recording session, guinea pigs were anesthetized
with a mixture of intramuscular xylazine hydrochloride (15 mg/kg) and ketamine
hydrochloride (35 mg/kg). The ABRs were measured using the Tucker-Davis Technology
System II (Tucker-Davis Technology, Gainsville, Fla) connected to a personal
computer (model E3110; Gateway, San Diego, Calif). Pure-tone stimuli were
digitally synthesized using SigGen software (Tucker-Davis Technology) and
presented through an earphone (Etymotic ER-2; Etymotic Research, Elk Grove
Village, Ill). Tone pips were presented at octave intervals from 500 to 16 000
Hz. Animals were presented with a stimulus intensity series, from a 90- to
10-dB sound pressure level (SPL) in 5-dB steps. Each ABR data point for an
individual animal consisted of an average of 500 stimulus presentations recorded
over a 10-millisecond period. Electrical activity was recorded via a platinum
needle electrode (Grass Telefactor, West Warwick, RI) inserted into the scalp
at the vertex, referenced to another needle electrode in a neck muscle. A
third needle ground electrode was placed at the tympanic bulla. The responses
at or near threshold were confirmed on a second testing. Threshold was defined
as the lowest intensity capable of producing a reproducible ABR waveform with
good morphology of wave III or V response. These ABR data were collected bilaterally
from all guinea pigs.
EXPERIMENTAL SURGERY
Prior to surgery guinea pigs were anesthetized with a combined mixture
of intramuscular xylazine hydrochloride (15 mg/kg) and ketamine hydrochloride
(35 mg/kg). Following a retroauriclular incision, tympanic bullae were opened
with a cutting bur. Middle ear lesions were bilateral in groups 1 and 2 and
unilateral in group 3. A 3 x 3-mm area of promontory ME mucosa close
to the round window niche was scraped away with a right angle elevator. Equivalent
volumes of isotonic sodium chloride-moistened packing material (ie, MeroGel
and AGS) were determined by side-by-side comparison as viewed using a dissecting
microscope and then each volume was subdivided for packing the MEs of animals
from groups 1 and 2. Packing material was placed in the posterior inferior
quadrant of each ME cavity. The packed area started anterior to the wound
margin on the promontory and anterosuperior to both the posterior crus of
the stapes and long process of the incus and continued until the opening of
the bulla. Thus, the packing material partially covered the promontory area
near the round window membrane niche, partially surrounded the posterior crus
of the stapes and long process of the incus, and completely filled the whole
round window membrane niche. Care was taken to ensure the continuity of the
ossicular chain and the integrity of the tympanic membrane in all animals.
To control for variations in surgical technique all procedures were performed
by only one of us (G.L.).
HISTOLOGIC FINDINGS
Six weeks after creating a lesion in ME mucosa, animals were deeply
anesthetized and transcardially perfused with 4% paraformaldehyde. Temporal
bones were harvested and tympanic bullae carefully opened. At postoperative
week 6, the amount of MeroGel and AGS packing material left in the ME was
evaluated, using the following system of scoring: 0, no packing material remained;
1, a membranelike sheet was present; and 2, a mass less than 20% of the original
packing material volume remained. Evaluation of the MEs was done using a surgical
microscope and was performed by the same observer (G.L.). Dissected temporal
bones were then fixed overnight in 4% paraformaldehyde at 4°C. The specimens
were transferred into a formic acid decalcification solution (Immunocal; Decal
Corp, Congers, NY) and were decalcified for 3 days at room temperature. Decalcified
temporal bones were dehydrated in increasing concentrations of ethyl alcohol
and subsequently embedded in paraffin. Seven-micrometer-thick serial sections
of the area of interest were cut, stained with toluidine blue, and inspected
using a light microscope (ZeissAxiophot; Carl Zeiss Microscope Systems, Oberkochen,
Germany).
STATISTICAL ANALYSIS
The  2 test was used to determine the level of statistical
significance between the amounts of MeroGel and AGS left in the ME cavities.
A t test was used to evaluate the level of significance
between ABR changes in the animals in all 3 groups. Statistical significance
was set at P<.05.
RESULTS
ABR RECORDINGS
For group 1 (unilateral MeroGel packing, bilateral wounding) a low-frequency
hearing loss (ie, 30- to 40-dB SPL) was noted in the MeroGel-packed ears at
postoperative day 5 when compared with the preoperative ABR thresholds. This
was statistically significant for 500-, 1000-, 2000-, and 4000-Hz pure-tone
stimuli (P<.001). Eighty percent of the guinea
pigs with MeroGel-packed ears (group 1) had a full recovery from these depressed
ABR thresholds by postoperative week 6 recovering to preoperative ABR threshold
levels of sensitivity (Figure 1A).
Twenty percent of the animals in group 1 had a partial recovery of the ABR
threshold (ie, 15- to 20-dB–SPL gain). There were no significant threshold
differences between preoperative and 6 weeks' postoperative ABRs for all frequencies
(all values P>.05).
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Figure 1. Hearing thresholds for pure tones
from 500 to 16 000 Hz are shown for the MeroGel-treated (an esterified
hyaluronic acid) (group 1) (A) and the absorbable gelatin sponge (AGS)–treated
(group 2) (B) guinea pigs. A comparison of gains in auditory brainstem recording
threshold sensitivities for the 500- to 4000-Hz range of frequency stimuli
at postoperative week 6 is presented for group 1 and 2 animals (C). SPL indicates
sound pressure level; single asterisk, P<.05;
double asterisks, P<.01; and triple asterisks, P<.001. See "Materials and Methods section for explanation
of groups.
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For group 2 (unilateral AGS packing, bilateral wounding) a statistically
significant low-frequency hearing loss (ie, 30- to 40-dB SPL) was also noted
in the AGS-packed ears at postoperative day 5 compared with preoperative ABR
thresholds, for 500-, 1000-, 2000-, and 4000-Hz pure-tone stimuli (all values P<.001). At postoperative week 6 the AGS-packed ears
showed some recovery of the hearing loss (ie, a 15- to 20-dB SPL gain), but
none of these ears recovered an ABR hearing threshold that was equivalent
to their preoperative levels of sensitivity (Figure 1B). There were statistically significant differences between
the low-frequency preoperative ABR hearing thresholds and the postoperative
6-week thresholds for group 2, for 500-, 1000-, 2000-, and 4000-Hz pure-tone
stimuli (P<.001). However, the gains in the ABR
hearing threshold between the low-frequency, postoperative 5-day ABR hearing
thresholds, and the postoperative 6-week ABR hearing thresholds for group
2 were sufficient to show statistical significance (all values P<.001).
All animals with packed MEs in group 1 (MeroGel) and group 2 (AGS) had
similar frequency ranges and hearing losses at postoperative day 5 (Figure 1, respectively). There was a significant
recovery of ABR hearing thresholds in both group 1 and group 2 animals at
postoperative week 6; however, a comparison of the ABR hearing thresholds
showed that the animals in group 1 had a greater improvement in their recovery
of ABR hearing thresholds than those in group 2, ie, P<.05
for 500 and 4000 Hz; P<.001 for 1000 Hz; and P<.01 for 2000 Hz (Figure 1C).
For groups 1 through 3 all nonpacked ears (ie, either wounded or unoperated
on) showed no significant changes in ABR hearing thresholds throughout the
course of this study (all values P>.05). In addition,
there were no observed high-frequency hearing deficits in any of the animals
tested, ie, 8000- to 16 000-Hz range, and the ultrahigh frequencies (ie,
>16 000 Hz) were not measured in this study.
ME MICROSCOPIC FINDINGS
Some microscopic residua of packing material was identifiable in the
MEs for both group 1 and 2 animals at postoperative week 6. Both the amount
and quality of the remaining ME-packing material differed greatly between
the animals in group 1 and group 2. A transparent, membranelike sheet was
found in 80% of MeroGel (group 1)-packed MEs at or near the wounding site
(Figure 2A). Only 2 of the group
1 MEs had a masslike residua of MeroGel that was less than 20% of the original
volume. In the AGS-packed MEs, a retained AGS mass was present in the ME of
every animal and the volume of this retained AGS ranged from more than 20%
to 50% of the original volume of packing material. All of the residual AGS-packing
material were localized to the wounding site where the original packing was
placed. Statistical analysis showed a significant difference (all values P<.01) in the amount of packing material remaining in
the ME at postoperative week 6 between the group 1 (MeroGel)- and group 2
(AGS-)treated animals (Table 1).
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Figure 2. A cross-section of the middle
ear cavity of a MeroGel-treated (an esterified hyaluronic acid) animal (group
1) at postoperative week 6. A, View of the membranelike structure (MR) and
normal-appearing mucosal membranes (MEM) (toluidine blue, original magnification
x62.5). RWM indicates round window membrane niche; ST, scala tympani.
B, View of the inflammatory cells (ICs) with the MR (toluidine blue, original
magnification x500).
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Evaluation of the Retention of Middle Ear Packing Material in Group
1 and Group 2 Animals at Postoperative Week 6
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ME HISTOLOGIC FINDINGS
No abnormalities were found in the MEs of any of the untreated animals
with lesions from groups 1 and 2 or in the control animals (group 3). The
histologic appearance of MeroGel-treated (group 1) MEs was characterized by
a membranelike sheet infiltrated with inflammatory cells that covered the
center of the area with the lesion(Figure
2). There was a slight increase in the thickness of the mucous membrane
at the wound site but there was no new bone formation in these ME cavities
(Figure 2A).
The AGS-treated (group 2) MEs retained a mass of AGS matrix in the area
of the lesion that surrounded the round window membrane niche (Figure 3A). These MEs all had inflammatory cells, marked new bone
formation (ie, either in the AGS matrix or attached to the wall of ME), new
vessel formation, and fibroblasts within the AGS matrix (Figure 3B). Normal healing of the lesion site was present in the
untreated ears with lesions (groups 1 through 3). Neither adhesions nor thickening
of the healing mucosa was observed in any of these control ears (Figure 4).
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Figure 3. A cross-section of the middle
ear cavity of an absorbable gelatin sponge (AGS)–treated animal at postoperative
week 6. A, View of residua of AGS filling of the middle ear cavity at the
lesion site (toluidine blue, original magnification x500). ST indicates
scala tympani; RWM, round window membrane; SV, scala vestibuli; and SM, scala
media. B, View of newly formed bone (NB) within the AGS residua (toluidine
blue, original magnification x62.5). C indicates bony wall of the cochlea.
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Figure 4. A cross-section of the middle
ear cavity (MEC) of an untreated (group 3) animal at postoperative week 6.
The MEC is free of debris. The mucosal membranes (MEM) appear normal (toluidine
blue, original magnification x125). BM indicates basilar membrane; ST,
scala tympani.
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All animals (groups 1 through 3) survived the duration of the experiment.
None of these animals exhibited ME or inner ear infections or balance disorders.
COMMENT
Supportive materials are commonly used in the ME as packing to enhance
healing of tympanic membrane repairs and to maintain the integrity of the
ME space during postsurgical wound healing. Hyaluronic acid is a naturally
occurring extracellular polysaccharide with a molecular weight in the range
of 2 to 4 million daltons.1 Although purified
HA has been shown to have a high level of tissue biocompatibility and to be
nontoxic to the inner ear,6, 7, 8
its viscous physical properties1 has limited
its use as a surgical packing material for the ME cavity. Because MeroGel
is an esterified form of HA, its physical properties are more like that of
a sponge, which make it an ideal packing material for ME application. Like
AGS, compressed MeroGel can be easily trimmed to allow for the insertion into
a surgical site within the ME cavity. When moistened with an isotonic sodium
chloride solution, MeroGel showed a high level of expandability that is a
necessary property for any supportive packing material to be used during ME
surgery. In this study, minimal residual MeroGel was encountered in the ME
cavity at postoperative week 6 in 8 of the 10 ME cavities examined. This resorption
period provides adequate time for support to aid in healing of ME tissues,
while limiting the amount of time the ME has to produce an inflammatory response.
Changes in auditory thresholds for pure-tone stimuli were evaluated
by ABR recordings which is a reliable test and allows for the evaluation of
the integrity of both the inner ear and MEs. Increases in auditory thresholds
for only the low frequencies in both the MeroGel- and AGS-treated ears at
postoperative day 5 indicate that both of these packing materials effect the
mobility of the ossicular chain and/or the round window and oval window membranes.
Recovery of hearing, therefore, most likely implies a recovery of the mobility
of the ossicular chain and/or of the round and oval window membranes. The
observed hearing recovery correlated with the disappearance of most of the
original volume of MeroGel packing and a lack of major tissue adhesions that
could compromise ossicular chain function. The histologic evaluation showed
scant adhesions in 20% of the animals and in 80% of the treated ears (group
1) that only a very small amount of the original MeroGel material (ie, membranelike
material) had remained in the ME cavity. In the AGS-packed ears, at postoperative
week 6, there was a persistence of a mass of AGS with adhesions in the ME
cavity and new bone deposition that resulted in a hearing loss that was most
probably of a conductive nature.
The ABR recordings also showed that MeroGel had no toxic effect on cochlear
hair cells based on the observation that there were no high-frequencies hearing
losses (ie, 8000-16 000 Hz) and that there was a total recovery of hearing
thresholds in 80% of the MeroGel-treated ears at postoperative week 6. Other
studies6, 8 also had a similar
result from the application of a viscous form of HA to the ME cavity of guinea
pigs and rats. Hearing thresholds of the HA-packed MEs were recovered to normal
levels in either 4 weeks or 3 months after HA application.
The comparison of MeroGel and AGS as packing materials for the ME is
limited by the fact that the experiment was terminated and analyzed at 6 weeks
after the placement of both materials in the ME cavity. A comparison of hearing
results between the MeroGel and AGS groups of animals at postoperative day
5 showed that the results at this stage were equivalent in groups 1 and 2,
respectively. Presumably, this hearing loss was of a conductive origin because
in both groups, the ME cavities were filled with packing material. Difference
in hearing results (ie, ABR thresholds) between groups 1 and 2 became significant
at postoperative week 6 (Figure 1C).
At this time, most of the animals that had their ears packed with MeroGel
had regained their baseline hearing sensitivity, while the AGS-treated animals
continued to exhibit a residual hearing impairment at low frequencies. This
hearing loss is significantly less severe than the hearing loss that the AGS-treated
animals had at postoperative day 5 (compare Figure 1A with 1B) demonstrating a partial recovery of hearing acuity
in the AGS-treated animals. There was no observable high-frequency hearing
loss in any of the animals and ultrahigh-frequency hearing, ie, above 16 000
Hz, was not measured in this study. Therefore, our conclusions about ototoxic
reactions are limited only to the tested frequencies, ie, 500 to 16 000
Hz.
When the MeroGel- and AGS-treated MEs were compared with surgical microscopic
and histologic evaluations, the following findings were noted: in the MeroGel-treated
ears, a thin transparent membranelike material was still present in the ME
at postoperative week 6 (Figure 2A).
This membranelike material showed some inflammatory cell infiltration (Figure 2B). In the AGS-treated group, the
ME still contained a mass of the material that covered the floor of the ME
and round window membrane niche area (Figure
3A). The mass of retained AGS was surrounded by inflammatory changes
and showed new bone formation (Figure 3B).
Most of the histologic studies in the literature have shown that use of the
viscous form of HA in the ME cavity has not caused either new bone or adhesions
to form in the ME cavity.6, 7 The
study11 evaluating the "foam" esterified form
of HA has reported new bone formation, mucosa thickness, and adhesions, although
they were moderate when compared with the adverse effects caused by the use
of AGS. The severity of pathologic change for both MeroGel and AGS might be
dependent on the severity and extent of ME mucosa wounding as well as the
position and amount of packing material applied within the ME cavity. Variations
in extent of wounding of the ME mucosa and amount and placement of packing
material may also have implications for the clinical application of the tested
packing materials. Therefore, the finding of the present study and of a previous
study11 both show a similar pattern in the
ME cavities response to esterified HA as compared with AGS-packing material
(Table 1 and Figure 1, Figure 2, and Figure 3).
New bone formation in the AGS-packed MEs may not necessarily correlate
with a similar reaction in human ears. The favorable results encountered in
the MeroGel-treated ears indicate that, in guinea pigs, MeroGel is a safe
packing material for applications in the ME cavity. Clinical studies will
be required to establish the clinical usefulness and safety of MeroGel as
a surgical packing material in the MEs of patients as an adjunct to aid healing
following ME surgery.
CONCLUSIONS
The results show that MeroGel is a nonototoxic substance that has a
high level of tissue biocompatibility with ME mucosa. It is an effective support
material for packing the ME during surgery and is easily expelled from the
ME cavity causing only a mild inflammatory response.
AUTHOR INFORMATION
Accepted for publication September 22, 2000.
This study was supported by a grant from the Carolina Ear Institute,
Raleigh, NC (Dr Van De Water).
Presented as a poster (abstract 31) at the 23rd Association for Resarch
in Otolaryngology Midwinter Research Meeting, St Petersburg Beach, Fla, February.
20-24, 2000.
From the Departments of Otolaryngology (Drs Li and Van De Water) and
Neuroscience (Dr Van De Water), Albert Einstein College of Medicine, Bronx,
NY; Department of Otolaryngology, Montefiore Medical Center, Bronx (Drs Feghali,
Dinces, and Van De Water); Department of Otolaryngology, Beth Israel Medical
Center, New York, NY (Dr Feghali); and Carolina Ear and Hearing Clinic, Raleigh,
NC (Dr McElveen).
Corresponding author and reprints: Thomas R. Van De Water, PhD, Department
of Otolaryngology, Albert Einstein College of Medicine, 1410 Pelham Pkwy S,
Kennedy Center, Room 302, Bronx, NY 10461 (e-mail: vandewat{at}aecom.yu.edu).
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