J Med Dent Sci 2011； 58： 113－121
Original Article
Epithelial regeneration after diffuse alveolar damage in relation to underlying
disease and DAD stage: an autopsy study
Satoru Asano1, 4), Tamiko Takemura2), Keiichi Katoh3), Masuzoh Taneda1) and Masanobu Kitagawa4)
1) Department of Intensive Care Unit, Japanese Red Cross Medical Center
2) Department of Pathology, Japanese Red Cross Medical Center
3) Department of Anesthesiology, Japanese Red Cross Medical Center
4) Department of Comprehensive Pathology, Aging and Developmental Sciences, Tokyo Medical and Dental
University, Graduate School
This study was conducted to characterize the
distribution of epithelial regeneration in lungs with
diffuse alveolar damage (DAD) and to ascertain
whether epithelial regeneration is associated with
the underlying disease and/or DAD stage. Methods:
Lung specimens obtained at autopsy from 18
patients were studied using p63, PE-10 and Ki-67
immunostaining. The patients were categorized
into two groups: patients with acute exacerbation
of idiopathic pulmonary fibrosis (AE-IPF; IPF group)
and patients without IPF (non-IPF group). Results:
p63-positive cells were mostly observed in the
alveolar ducts. No significant difference in the
appearance of the p63-positive cells was observed
between the IPF and non-IPF groups or between
the exudative and the proliferative stages. PE-10positive cells were observed in the alveoli. The
number of PE-10-positive cells was significantly
higher during the proliferative stage than during
the exudative stage. However, p63-positive cells
and PE-10-positive cells appeared at day 6 in the
earliest case during the exudative stage and Ki67-positive cells were observed during the
exudative stage. Conclusion: Epithelial
regeneration was not associated with the
underlying disease. This study revealed that
regenerative epithelial cells appeared at day 6
Corresponding Author: Satoru Asano
Department of Intensive Care Unit, Japanese Red Cross Medical
Center Shibuya-ku Hiroo 4-1-22, Tokyo, Japan, 150-8935
Tel: +81-3-3400-1311　Fax: +81-3-3409-1604
E-mail: [email protected]
Received May 25；Accepted September 9, 2011
during the exudative stage and that the number of
type Ⅱ pneumocytes increased significantly during
the proliferative stage.
Key words: d i f f u s e a l v e o l a r d a m a g e ; e p i t h e l i a l
regeneration; p63; bronchiolar basal cells;
type Ⅱ pneumocytes
Introduction
Diffuse alveolar damage (DAD) is a common
pathological feature of acute lung injury (ALI) and acute
respiratory distress syndrome (ARDS). DAD is still
difficult to treat clinically because the development of
DAD is multifactorial, with immunodeficiency, infections
and idiopathic pulmonary fibrosis all being potential
causes. DAD is an extremely treatment-resistant
condition, and no effective treatment has yet been
established.
The fundamental pathological features of DAD involve
alveolar epithelial and endothelial damage, resulting in
the sloughing of the alveolar lining cells, the denudation
of the epithelial basement membrane and hyaline
1
membrane (HM) formation. Therefore, epithelial cells
observed at autopsy are thought to be regenerative
cells or cells avoided injury. DAD progresses from an
early exudative stage, with the destruction of alveolar
structures and the formation of a characteristic HM, to
a late proliferative stage with architectural remodeling,
leading to fibrosis and the simplification of the
pulmonary architecture. 1
Since the most problematic features of DAD are
the dysf unct ional repair pr oces ses, norm al re epithelialization and remodeling of the microvasculature
114
S. Asano et al.
are thought to be important for preserving respiratory
function in patients with DAD. 2,3 However, in clinical
settings, patients with DAD cannot wait until the
damaged lung is correctly repaired, because the initial
insult is too severe for the renewal of normal epithelial
cells to occur fast enough. Any delay in the repair
processes caused by the abnormal responses of other
cells interacting with the epithelium may exert an
adverse effect on normal repair.
Generally, the progenitor stem cells involved in lung
development and regeneration are thought to be the
basal cells of respiratory bronchioles. 4 However, the
behavior of bronchiolar basal cells in lungs with DAD is
not clear based on morphological evaluations using
hematoxylin and eosin (HE) staining. These cells can be
highlighted using p63 immunostaining. 4 p63 plays an
important role in the physiological functions of basal
cells and is constitutively expressed in stratified basal
cells. An analysis of p63 is particularly informative for
detecting the involvement of basal cells in epithelial
repair and tissue remodeling in DAD. 4,5 p63-positive
cells usually appear as bronchiolar basal cell and do
not appear in other areas in normal lung tissue. p63positive cells observed away from respiratory
bronchioles at the time of autopsy are thought to be
regenerative bronchiolar basal cells.
On the other hand, type Ⅱ pneumocytes differentiate
into type Ⅰ cells and are associated with the
reconstruction of the normal alveolar architecture. 6
Anti-human surfactant protein A (PE-10) is an indicator
reflecting the localization and development of
pulmonary surfactant by type Ⅱ pneumocytes. 7,8
Ki-67 is associated with cellular proliferation. Ki-67
positive cells are present during all active phases of the
cell cycle but are absent from resting cells. 9,10 The
observation of Ki-67-positive cells provides information
about which part of the DAD-affected lung is highly
involved in proliferation.
In t he present stu d y , we ex a min e d ep i t helial
regeneration in lungs with DAD in relation to the
underlying disease and also to the DAD stage using
light microscopy and immunohistochemical staining for
p63, PE-10 and Ki-67.
Materials and Methods
Patient selection
Sixty-six patients treated in the intensive care unit
(ICU) of the Japanese Red Cross Medical Center
between 1998 and 2010 were autopsied. Fifty-three of
the 66 cases had died of ALI/ARDS. DAD was
J Med Dent Sci
histopathologically detected in 18 of these 53 (33.9%)
autopsied cases. These 18 patients were included in
the present study. Out of the 18 patients, 9 suffered
from acute exacerbation of idiopathic pulmonary
fibrosis (AE-IPF), i.e., the idiopathic pulmonary fibrosis
(IPF) group. The others constituted the non-IPF group.
When desaturation was observed, the patients were
transferred to the ICU for respiratory management and
were immediately intubated. Desaturation was
considered to be the onset of ALI/ARDS.
Informed consent to participate in this study was
obtained from all the patients’ families. This study was
approved by our institutional review board (approval
number 2).
Tissue preparation
The lungs were fixed using the transbronchial infusion
of 10% formalin at a pressure of 25 cmH2O. More than
one section was cut from each lobe of the lungs for
histological examination (the average number of
sections was 20). We selected one representative
section of DAD. For the cases in the IPF group, we
selected a functioning area that was located away from
the sites of previous structural remodeling, such as
honeycombing. In the non-IPF group, we selected an
area that was located away from infectious areas.
These areas were considered to be responsible for the
respiratory failure. 4μm-thick paraffin sections were
cut and stained with hematoxylin and eosin, elastic van
Gieson, Masson’s trichrome and periodic acid Schiff
(PAS). We then examined the localization of the
regenerating epithelium, pneumocyte hyperplasia, and
squamous metaplasia.
The immunohistochemical staining of formalin-fixed,
paraffin-embedded sections was performed using p63
(Dako; diluted 1:50), PE-10 (Dako; diluted 1:40) and Ki67 (Dako; diluted 1:50). All the procedures were
performed using a Ventana auto-immunostainer
(Ventana Medical Systems, Inc., USA).
We analyzed the p63-positive cells around the
respiratory bronchioles, except for the p63-positive
basal cells of the bronchioles, and counted the total
number of p63-positive cells. The score was considered
positive only if distinct nuclear staining was present (-,
absent; +, positive), since the number of p63-positive
cells was relatively small.
In each case, PE-10-positive cells were examined in
the alveoli of 4 lesions containing cells that were
positive for p63; the examinations were performed
under a magnification of × 40 with an objective lens.
The score was based on the degree of positivity (low,
115
Epithelial regeneration after DAD
Table 1. Patients demographics
Case no
Age / Sex
Underlying disease
Duration (d)
DAD stage
1
66 / M
AE-IPF
6
Exudative
PaO2/FIO2 (%)
79
2
59 / M
AE-IPF
6
Exudative
122
103
3
65 / M
AE-IPF
6
Exudative
4
76 / M
AE-IPF
8
Exudative
93
5
56 / M
AE-IPF
11
Proliferative
76
6
81 / M
AE-IPF
17
Proliferative
62
7
76 / M
AE-IPF
17
Proliferative
71
8
81 / M
AE-IPF
21
Proliferative
128
9
62 / F
AE-IPF
28
Proliferative
49
mean ± SD
10
69 / M
13.3 ± 7.8
AML
87.3 ± 26.6
6
Exudative
138
149
11
39 / F
MDS
8
Proliferative
12
26 / M
Hemophilia A
10
Exudative
90
13
77 / F
Aspiration pneumonia
12
Exudative
116
14
41 / F
MM
14
Proliferative
157
15
40 / M
Lennert’s lymphoma
14
Proliferative
302
16
71 / M
Aspiration pneumonia
19
Proliferative
116
17
91 / F
Aspiration pneumonia
30
Proliferative
47
18
57 / M
DCM
31
Proliferative
89
mean ± SD
16.0 ± 9.0
134.0 ± 71.3
Duration: duration between onset and death.
AE-IPF: acute exacerbation of idiopathic pulmonary fibrosis. DAD: diffuse alveolar damage.
MM: multiple myeloma. AML: acute myelogenous leukemia.
MDS: myelodysplasic syndrome. DCM: dilated cardiomyopathy.
0% ~ 50% positive immunoreactivity; high, 50% ~ 100%
positive immunoreactivity). The percent immunoreactivity
was determined based on the total number of PE-10positive cells / the total number of epithelia × 100.
Ki-67-positive cells were examined and counted in
the respiratory bronchioles, the alveolar duct, and the
alveoli to determine the percent positivity among 100
epithelial cells under a magnification of × 40 with an
objective lens. The score was the percentage of Ki-67positive cells.
Statistical analysis
Pathological data were analyzed statistically using a
Fisher exact test for the p63-positive cells, the chisquare test for the score of PE-10-positive cells, and an
unpaired Student t -test for the percentage of Ki-67positive cells between the IPF and non-IPF groups of
DAD, and also between the exudative and proliferative
stages of DAD. The distribution values were presented
as the means ± SD. A P value of 0.05 was used as the
cut-off point for statistical significance.
Results
Clinical characteristics
Demographic data including age, sex, duration of
mechanical ventilation from onset until death and
underlying diseases are presented in Table 1. Out of
the 18 patients, 9 suffered from AE-IPF, i.e., the IPF
group. The non-IPF group consisted of patients with
hematological disorders (n = 5), aspiration pneumonia
(n = 3) and dilated cardiomyopathy (n = 1).
The average age of the study population was 62.9 ±
17.3 (26 - 91) years. There were 13 men and 5 women.
The average time interval from onset of DAD until death
was 14.7 ± 8.4 days. The interval was 13.3 ± 7.8 days
in the IPF group and 16.0 ± 9.0 days in the non-IPF
group. No significant difference in the interval was
noted between the IPF and non-IPF groups (P = 0.51).
The ARDS network has reported that a lower tidal
volume (6mL/kg) is appropriate for mechanical
ventilation in patients with ALI/ARDS. 11 Patients had
received mechanical ventilation using synchronized
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S. Asano et al.
intermittent mandatory ventilation (SIMV) with a positive
end expiratory pressure (PEEP) and pressure support
(PS). The fractional concentration of oxygen in the
inspired air (FIO2) had reached 1.0 in the patients by the
time of death. The ratio of the arterial oxygen partial
pressure (PaO2) to FIO2 was used to define the severity
of ALI/ARDS, so as to adjust for the ventilation settings.
The PaO2/FIO2 ratio just after intubation was 111.1 ±
57.7 (47 - 302). The ratio of PaO2 to FIO2 was 87.3 ±
26.6 in the IPF group and 134.0 ± 71.3 in the non-IPF
group. No significant difference in the ratio was
observed between the IPF and non-IPF groups (P =
0.08). Seventeen of the 18 patients met the criteria of
the Clinical Practical Guidelines for ALI and ARDS
syndrome . 12
Histological findings
The lungs of 7 of 18 patients showed evidence of an
exudative stage, with interstitial edema and hyaline
membranes. The lungs of 11 patients showed signs of a
proliferative and organizing stage, with interstitial
inflammation, fibroblastic proliferation and dilatation of
the alveolar ducts (Table 1).
Four of the 9 cases in the IPF group exhibited an
exudative stage. The other 5 cases exhibited a
proliferative stage. Three of the 9 cases in the non-IPF
group exhibited an exudative stage, while 6 exhibited a
proliferative stage.
Alveolar pneumocyte hyperplasia was observed
around the alveolar ducts in 13 of the 18 cases (72.2%).
In the IPF group, alveolar pneumocyte hyperplasia was
seen in 6 of the 9 cases (66.7%). In the non-IPF group,
however, alveolar pneumocyte hyperplasia was seen in
7 of the 9 cases (77.8%) (Table 2).
Squamous metaplasia was found around the
respiratory bronchioles in 14 of the 18 cases (77.8%).
In the IPF group, squamous metaplasia was observed in
9 cases (100%). In the non-IPF group, squamous
metaplasia was seen in 5 of the 9 cases (55.6%) (Table
2).
None of the cases had cytomegalic inclusions.
Table 2. C
omparison of data between the IPF group and the nonIPF group
Squamous metaplasia
Hyperplasia
IPF group（n=9）
9（100%）
6（66.7%）
non-IPF group（n=9）
5（55.6%）
7（77.8%）
Hyperplasia: alveolar pneumocyte hyperplasia.
IPF: idiopathic pulmonary fibrosis.
J Med Dent Sci
Immunohistochemistry
Regenerative epithelia were positive for p63, and the
expression of p63 was frequently observed in areas of
squamous metaplasia (Figure 1). p63-positive cells were
observed close to the respiratory bronchioles in 8 of
the 9 IPF cases (88.9%) but in 5 of the 9 non-IPF cases
(55.6%). p63-positive cells were detected in the
alveolar ducts in 6 cases (67.0%) in the IPF group and
in 3 cases (33.3%) in the non-IPF group. No significant
difference in the appearance of p63 was observed
between the IPF and non-IPF groups (Table 3).
In the exudative stage, p63-positive cells were
observed close to the respiratory bronchioles in 3 of
the 7 cases (33.3%) and in the alveolar ducts in 2 of
the 7 cases (28.6%). In the proliferative stage, p63positive cells were observed close to the respiratory
bronchioles in 9 of the 11 cases (81.8%) and in 7 of the
11 cases (63.6%) in the alveolar ducts. No significant
difference in the appearance of p63 was observed
between the exudative and proliferative stages (Table
4).
p63-positive cells were not observed in the alveoli.
p63-positive cells were observed in the alveolar duct at
6 days after the onset of ALI/ARDS in the earliest case
during the exudative stage (Case number 3) (Tables 1,
4).
PE-10-positive cells in this study were regenerative
type Ⅱ pneumocytes. Furthermore, hyaline membrane
and surfactant apoprotein were also detected using PE10 immunostaining. Occasionally, some swollen
pneumocytes that resembled regenerative epithelial
cells on HE-stained specimens did not exhibit PE-10
(Figure 2). The score for PE-10-positive cells did not
differ significantly between the IPF and non-IPF groups
(Table 3). However, the number of type Ⅱ pneumocytes
was higher during the proliferative stage, compared
with the exudative stage (Figure 2) and a significant
difference in the appearance of the type Ⅱ
pneumocytes was observed between the exudative and
proliferative stages (P = 0.013) (Table 4).
On the other hand, PE-10-positive cells appeared in
the alveoli at 6 days after the onset of ALI/ARDS in the
earliest case during the exudative stage (Case number
10) (Tables 1, 4).
Ki-67-positive cells were sporadically seen at sites of
squamous metaplasia (Figure 3). The percentage of Ki67-positive cells was 6.0% ± 5.7% in the IPF group and
4.5% ± 5.1% in the non-IPF group (Table 3). During the
exudative stage, the percentage of Ki-67-positive cells
was 5.9% ± 5.8%, but the percentage was 4.3% ±
4.8% during the proliferative stage (Table 4). Ki-67
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Epithelial regeneration after DAD
Table 3. E
xpressions of regenerative epithelium in underlying diseases
p63
PE-10
Ki-67
Respiratory bronchiole
Alveolar duct
low
high
positive
IPF group (n = 9)
8 (88.9%)
6 (67%)
6
3
6.0 ± 5.7%
non-IPF group (n =9 )
5 (55.6%)
3 (33.3%)
4
5
4.5 ± 5.1%
P value
0.29
0.35
0.18
0.63
The data are presented in numbers and percentages.
IPF: idiopathic pulmonary fibrosis.
low: 0% ～ 50% positive immunoreactivity. high: 50% ～ 100% positive immunoreactivity.
Table 4. E
xpressions of regenerative epithelium in DAD stage
p63
PE-10
Ki-67
Respiratory bronchiole
Alveolar duct
low
high
positive
Exudative stage (n = 7)
3 (33.3%)
2 (28.6%)
6
1
5.9 ± 5.8%
Proliferative stage (n =11)
9 (81.8%)
7 (63.6%)
4
7
4.3 ± 4.8%
P value
0.14
0.33
0.013
0.6
The data are presented in numbers and percentages.
IPF: idiopathic pulmonary fibrosis.
low: 0% ～ 50% positive immunoreactivity. high: 50% ～ 100% positive immunoreactivity.
expression did no differ between the IPF and non-IPF
groups, and no significant difference was observed
between the exudative stage and the proliferative
stage. Ki-67-positive cells were observed mostly in the
respiratory bronchioles and alveolar ducts. In the
alveoli, because the pulmonary structure was severely
distorted, the proliferative cells were difficult to
discriminate from lymphocytes, which also stained
positive for Ki-67 (Figure 4).
Discussion
The mechanism of epithelial regeneration in DAD is
complex, involving several processes including acute
inflammation, regeneration, proliferation, remodeling
and fibrosis. 3 In this study, we focused on epithelial
regeneration after DAD in view of the relationship with
the underlying disease and the time interval from the
onset of DAD until death. Morphological behavior has
not yet been fully investigated in terms of tissue repair
in patients with DAD. Also, the participation of
bronchiolar basal cells and type Ⅱ pneumocytes in the
regenerative process in lungs with DAD has not been
reported.
Alveolar pneumocyte hyperplasia is characterized by
the proliferation of spaced cuboidal cells along the
alveolar septa and is often characterized by the
swelling of the cytoplasm and nuclei, compared with
normal flat type Ⅱ pneumocytes. These cells never
appear in normal lung. The epithelial cells observed in
autopsied lungs with DAD are thought to be
regenerative cells, most of which are hyperplastic. 1
Hyperplastic pneumocytes can be regarded as part of
the physiological response to alveolar damage, because
type Ⅱ pneumocytes are seen in a variety of conditions
in which alveolar proliferation or regeneration occurs. 1
Squamous metaplasia is observed in 5% of patients
with IPF. 13 The squamous metaplasia observed in this
study was thought to be newly caused by acute lung
injury, since areas distant from previously IPF-involved
areas were examined. Since 100% of the cases in the
IPF group but 55.6% of the cases in the non-IPF group
exhibited squamous metaplasia, these results suggest
that patients with IPF may also have an increased
susceptibility to acute lung injury in response to a
variety of factors. However, this type of response
would also trigger squamous metaplasia or pneumocyte
hyperplasia as an exaggerated repair process. In
addition, patients with ALI/ARDS placed on mechanical
ventilators would experience mechanical distention of
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S. Asano et al.
J Med Dent Sci
（a）
（b）
Figure 1 : (a) p63-positive cells are seen in the exudative stage of IPF case (HE & p63, original magnification × 20).
(b) p63-positive cells are observed in the proliferative stage of non-IPF case (HE & p63, original magnification × 20).
the alveolar ducts, possibly contributing to further
injury or adversely affecting the repair mechanisms. 3
p63-positive cells are considered to be progenitor
cells during epithelial repair. 4 When the lung sustains an
injurious insult, the basal cells are regulated to function
as stem cells, promoting tissue repair and epithelial
regeneration. We speculated that bronchiolar basal
cells might expand to contribute to the structural repair
close to the respiratory bronchioles and injured alveoli
in a chronological fashion. On the other hand, in nonIPF cases, residual progenitor stem cells are directly
recruited to the injured area and extend over the
alveolar surface, with the proliferation and phenotypic
differentiation of these progenitor cells leading to the
recovery of epithelial function. 3
In this study, p63-positive cells were observed close
to the respiratory bronchioles and in the alveolar ducts
from the exudative stage to the proliferative stage and
were considered to be regenerative bronchiolar basal
cells. No statistically significant difference was
observed among the DAD stages (Table 4). However, in
the exudative stage, regenerative bronchiolar basal
cells in the alveolar duct were observed at 6 days after
the onset of ALI/ARDS (Case number 3) and Ki-67positive cells were observed in 5.9 ± 5.8% (Tables 1,
4). Regenerative bronchiolar basal cells are thought to
appear in the exudative stage, even while hyaline
membrane formation remains prominent.
As for PE-10-positive cells, Kawanami reported
that type Ⅱ pneumocytes were activated in areas
with less severe degrees of fibrosis; 14 in this study,
however, no significant difference between the IPF
Epithelial regeneration after DAD
119
（a）
（b）
Figure 2 : (a) P
E-10-positive cells are not detected in the alveolus in the exudative stage of IPF case (HE & PE-10, original
magnification × 40).
(b) PE-10-positive cells are strongly positive in the alveolus in the proliferative stage of IPF case. Some cells are negative
for PE-10 (HE & PE-10, original magnification × 40).
group and the non-IPF group was observed (Table 3).
On the other hand, type Ⅱ pneumocytes are known
to increase during the proliferative stage. 15 Regarding
immunoreactivity according to the stage of DAD,
our data also revealed that type Ⅱ pneumocytes
were significantly activated during the proliferative
stage, compared with the exudative stage (Table 4).
Two crucial features of the remodeling process are
migration and the proliferation of epithelial cells, which
rapidly restores and reseals the denuded alveolar
surface. 3 Type Ⅱ pneumocytes are thought to be the
precursors of type Ⅰ cells. These cells differentiate and
transform into type Ⅰ cells, replacing the sloughed type
Ⅰ cells and covering the denuded basal membrane
during normal epithelial repair. 6,16 The remodeling of the
pulmonary architecture is thought to be promoted by
the proliferation of typeⅡ pneumocytes.
In this study, however, PE-10-positive cells were
observed in the exudative stage at 6 days after DAD in
one case (Case number 10), and Ki-67-positive cells
were observed in 5.9 ± 5.8% (Tables 1, 4). Since PE10-positive cells are regenerative type Ⅱ pneumocytes,
regenerative type Ⅱ pneumocytes were considered to
begin to proliferate during the exudative stage. PE-10
staining along with HE staining is useful for detecting
the stage of DAD, although some pneumocytes were
negative for PE-10 (Figure 2).
The percentage of Ki-67-positive cells did not differ
significantly between the IPF group and the non-IPF
group, and between the exudative stage and the
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S. Asano et al.
J Med Dent Sci
Figure 3 : K
i-67-positive cells are observed at 17 days in squamous metaplasia in the proliferative stage of IPF case (HE & Ki-67,
original magnification × 40).
（a）
（b）
Figure 4 : (a) Ki-67-positive cells are observed in alveolus in the exudative stage of IPF case (HE & Ki-67, original magnification × 40).
(b) Ki-67-positive cells are seen in alveolus in the proliferative stage of IPF case (HE & Ki-67, original magnification × 40).
Epithelial regeneration after DAD
proliferative stage. However, Ki-67-positive cells were
already observed from the exudative stage (Table 4).
Since Katzenstein mentioned that alveolar lining cell
hyperplasia occurs 3 to 7 days after injury, 1 the
appearance of Ki-67-positive cells during the exudative
stage is reasonable.
Conclusions
This study characterized epithelial regeneration in
lungs with DAD using immunostaining for p63, PE-10
and Ki-67. Regenerative bronchiolar basal cells did not
differ significantly between the IPF group and the nonIPF group or between the exudative stage and the
proliferative stage. Regenerative type Ⅱ pneumocytes
increased significantly during the proliferative stage
compared with the exudative stage. On the other hand,
it was observed in this study that regenerative
bronchiolar basal cells and regenerative type Ⅱ
pneumocytes appeared at 6 days after the onset of
ALI/ARDS in the earliest case during the exudative
stage of DAD. Further investigation is needed to identify
a treatment strategy to promote the epithelial
regeneration after DAD.
1.
2.
3.
4.
5.
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