Abnormal Primary Tissue Collagen Composition in the Skin of Recurrent Incisional Hernia Patients
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Abnormal Primary Tissue Collagen Composition
in the Skin of Recurrent Incisional
Hernia Patients
BRENT WHITE, M.D., CHARLES OSIER, B.S., NANA GLETSU, PH.D., LOUIS JEANSONNE, M.D.,
MERCEDEH BAGHAI, M.D., MELANIE SHERMAN, PH.D., C DANIEL SMITH, M.D., BRUCE RAMSHAW, M.D.,
EDWARD LIN, D.O.
From the Hernia Institute, Emory Endosurgery Unit, Department of Surgery, Emory University
School of Medicine, Atlanta, Georgia
Recurrence of incisiotial hernia may be as high as 50 per cent. Abnormal collagen I/III ratios have
been observed within scar tissue of patients with recurrent incisional hernias. We sought to
determine whether collagen composition in primary, nonscarred tissue was similarly affected in
these patients. In this prospective, case-control study, nonscarred, primary abdominal wall skin
and fascia biopsies were obtained in 12 patients with a history of recurrent incisional hernias and
11 control subjects without any history of hernia while undergoing abdominal laparoscopic
surgery. Tissue protein expression of collagen I and III was assessed by immunohistochemistry
followed by densitometry analysis. The collagen I/III ratio in skin biopsies from the recurrent
hernia group was significantly less compared with control subjects (0.88 ± 0.01 versus 0.98 ± 0.04,
respectively, P < 0.05). Fascia biopsies from patients with recurrent hernias was not significantly
decreased in collagen I/III ratio compared with control subjects (0.90 ± 0.04 versus 0.94 ± 0.03,
respectively, P = 0.17). Decreased collagen I/III ratios within the skin of patients with recurrent
hernias not involved with scar or healing tissue suggest an underlying collagen composition
defect. Such a primary collagen defect, in addition to abnormal scar formation, likely plays a
significant role in the pathogenesis of recurrent incisional hernias.
I NCISIONAL HERNIAS CAN BE A major Complication of
the 9.8 million abdominal operations that are per-
formed in the United States each year.' Estimates of
composed of three polypeptide chains that intertwine
to form a triple helix structure The variation in chain
combinations account for different collagen molecules
failed primary fascial closure after laparotomy have (collagen types I, TI. III. and so on, reviewed in Van-
been reported to be as high as 50 per cent.- Abdominal der-Rest et al.'^). The collagen I/ill ratio of any given
incisional hernias can subsequently lead to chronic tissue determines its tensile strength and mechanical
pain, bowel obstruction, or incarceration. Based on stability with an increase in collagen type HI leading to
research by Luijendink et a!., up to 58 per cent of abnormal crosslinking of fibrils.'' '^^ This abnomial
nonmesh incisional hernia repairs will recur.^ Such a crosslinking ultimately reduces the tensile strength of
high recurrence rate has not only emphasized the im- the abdominal wall, making it more susceptible to her-
portance of tnesh repairs and continued reseaich in nia formation under increased stress.
development of better mesh material, but also has After injury, balanced collagen maturation and deg-
prompted further research on primary tissue healing radation is a requirement for normal scar formation.
mechanisms and physiology of scar formation. Prior research has focused on the pathogenesis of in-
Collagen is the principal component of the extracel- cisional hernia as a disorder of collagen regeneration
lular matrix of both ptimary and scar tissue."* It is during wound healing. Specifically, decreases in the
collagen 1/IIi ratio in scar tissue from both skin and
fascial sites have been demonstrated in several studies
using both protein and niRNA assays when comparing
Address correspondence and reprint requests to Edward Lin, patients with hernias with nomial subjects.-- '^- "• '^ To
D,0., Assistant Professor of Surgery. Departmenl of Surgery, date, comparative analysis of the collagen content of
Emory University School of Medicine, 1364 Clifton Road. H124,
Atlanta, GA 30322, E-mail: elin2@eniory.edu. primary, nonscarred connective tissue taken from pa-
This research was supported in part by a research grant from tients with recurrent incisional hernias and healthy
W,L. Gore & Associates. control subjects has yet to be performed.
1254
No. 12 ABNORMAL PRIMARY TISSUE COLLAGEN OOMPOSITION White et at. 1255
In this prospective, case-control study, we assessed were performed at room temperature and sections
collagLMi biochemistry in abdominal wall fascia and were washed with Tris-bulfered saline buffer between
skin biopsies obtained from patients with recurreni incubations. Coverslipping was performed using the
incisional hernias and from a control group (no history Tissue-Tek SCA (Sakura Finetek USA. Inc.. Torrance,
of incisional hernias). Taking full advantage of lapa- CA) automatic coverslipper. After staining, regions of
roscopic surgical techniques, we focused on non- skin (subepidermal) and fascia were imaged and cap-
scarred tissue located away from prior incisional sites tured in a high-power field (400x) by a digital camera
to determine if there were primary defects in skin and (Nikon Coolpix 4500. Tokyo. Japan) using Slidebook
fascial collagen that were not confounded by previous software (Intelligent Imaging innovations. Denver.
healing. Our hypothesis was that patients with recur- CO) based on a Zeiss Axiovert 200M inverted micro-
rent incisional hernias had inherent alterations in col- scope (Carl Zeiss, Thornwood, NY).
lagen I and III deposition in skin and fascia compared
with a group of patients with normal wound healing. Densitometry
Labeled collagen types I and III were quantified
Materials and Methods through densitometry measurements using ImageJ
Patients and Specimens software (National Institutes of Health. Bethesda,
MD).'** The entire image was selected for each tissue
A total ot" 32 patients consented to participate in this and converted to a 32-bit gray scale image (gray =
study. Informed consent was obtained as approved by 0,299 red -f- 0.587 green + 0).'^ The image was then
the institution's Institutional Review Board. Five pa- inverted to display labeled tissue as bright (Fig. I).
tients were excluded for known connective tissue dis- The mean gray value (the sum of the gray values of all
order, if they were undergoing initial (nonrecurrent) the pixels in the selection divided by the number of
hernia repair, or if they were undergoing ongoing ste- pixels) was reported in calibrated units of opticai den-
roid therapy. Four patients cancelled their surgical sity.
procedure. All patient medical records were reviewed
for demographic data collection and to obtain relevant
surgical and medical history. Statistical Analysis
During each patient's operation, a small piece of Data was analyzed using R statistical software (R
elliptical nonscarred skin was sharply excised at the Foundation, Vienna. Austria). The ratio of collagen I
site of a lateral trocar incision located well away from to collagen III density was computed for each skin and
the site of previous surgery and scar tissue. Subse- fascia specimen. Mann-Whitney tests were used to
quently, a small piece of transversalis fascia was compare the collagen 1/collagen III ratios between pa-
sharply excised under direct laparoscopic visualization tient groups for both skin and fascia. Data is expressed
well away from any scar, hernia defects, or other pa- as mean ± standard etror of mean. The significance
thology. Fascia and skin specimens were then dis- level was set at P < 0.05.
sected free from surrounding adipose tissue and im- Subanalysis was then performed comparing the ex-
mediately fixed in 10 per cent formaldehyde. perimental group witb the control patients who had
undergone prior abdominal surgery without hernia for-
hnnmnohistocliemistry and Microscopy mation. Mann-Whitney tests were again used to com-
pare the mean collagen I/III ratios between patient
Specimens were then embedded in paraffin, cut to groups for both skin and fascia. Data is expressed as
5-(jLm-thick sections, and adhered to a slide. The slides mean ± standard error of mean. The significance level
were then deparaffinized and rehydrated. Antigen re- was set at P < 0.05.
trieval was in a citrate buffer (pH 6) using an electric
pressure cooker for 5 minutes at 120°C with cooling
for 10 minutes before immunostaining. All tissues Results
were then exposed to 3 per cent hydrogen peroxide for Twenty-three patients were included in the final
5 minutes, collagen antibodies for 30 minutes, labeled study analysis and divided into two categories. The
polymer, horseradish peroxidase for 30 minutes, di- study group consisted of patients with a history of
aminobenzidine as chromogen for 5 minutes, and au- recurrent incisional hernia undergoing surgery (n =
tomation hematoxylin (Dako, Carpinteria. CA) as 12). The control group consisted of patients who had
counterstain for \5 minutes. The primary antibody no clinically evident or historical incisional hernia
used was a commercial peroxidase conjugated, goat (n ^ II), Demographic data, including age, gender,
anti-human IgG for collagen types I and III (Santa body mass index, and relevant surgical and medical
Cruz Biotechnology. Santa Cruz, CA). Incubations history, is noted in Table 1. There were no statistically
1256 THE AMERICAN SURGEON December 2007 Vol. 73
evident on a microscopic level (Fig. I). Analysis of
optical density collagen I/III ratios in skin and fascia
samples found that skin biopsies obtained from pa-
tients with hernias had significantly lower collagen
I/III ratios compared with skin biopsies obtained from
control subjects (0.88 ± 0.01 versus 0.98 ± 0.04, P <
0.05, Fig. 2). When comparing collagen I/TTl ratios iti
fascial samples biopsied from patients with hernias
versus control subjects, lower ratios were found in
samples obtained from patients with hernias, although
this difference was not statistically significant (0.90 ±
0.04 versus 0.94 ± 0.03, P = 0.17, Fig. 2).
Subanalysis of the experimental group (n — 12)
compared with the control subjects who had under-
gone prior abdominal surgery without hernia forma-
tion (n — 6) did not alter the findings noted in our
main analysis or their significance for skin (0.88 ±
0.01 versus 1.00 ± 0.05, P = 0.02) or fascia (0.90 ±
0.04 versus 0.94 ± 0.03, P = 0.25).
FIG. 1, IgG labeling of collagen types I and III in skin (400x. Discussion
white is collagen). Note the decrease in collagen type I (A) and
increase In collagen type III (B) in the tissue from a patient with a This study shows a decreased collagen I/III ratio in
history of recurrenl incisional hernia compared wilh control sub- primary skin and fascia biopsies of patients with re-
jects (C, collagen type 1. D. collagen type III). current incisional hernias when compared with their
normal counterparts. Skin samples obtained from pa-
TABLE 1. Patient Demographics tients with hernias had significantly lower collagen
Variable Control Hernia I/III ratios when compared with control subjects. Al-
though not significant, fascia biopsies obtained from
Number 11 12
Men
patients with hernias versus control subjects also had a
4 2
WotnetT 7 10 lower collagen I/III ratio.
Age (years) In normal tissue, collagen I and collagen II maintain
Mean 51.4 50 a relatively constant ratio. Previous studies have dem-
Range 30-74 37-62
Body mass onstrated abnormalities in the ratio of these collagen
index (kg/m^) molecules in patients with a number of genetic condi-
Mean 35.4 34.5 tions known to predispose to hernia formation such as
Range 22-53 23^7
Prior abdominal
surgery 1.06
Mean 1.3 2.6
Range (M 1-8
Prior hernia
repair
Mean 0 4.7
Range — 2-23
Diabetes
mellitus 2 2
Smoke tobacco 1 3
significant differences in age, body mass index, gen-
der, diabetes, or tobacco use in control subjects versus
patients with hernias. As expected, patients in the ex- Control Hernia Control Hernia
perimental group with recurrent incisional hernias had Skin Tissue Fascial Tissue
experienced more abdominal surgery compared with FIG, 2. Subepidermal (skin) and transversalis fascia eollagcn
control subjects {P < 0.05). i/lll ratio (ratio of optical density of eoltagen lype 1 to optical
density of collagen type III in a high-power field). The data arc
Differences in collagen type staining between con- presented as mean values wilh standard error, {*P < 0.05 versus
trol subjects and patients with hernias were visually control).
No. 12 ABNORMAL PRIMARY TISSUE COLLAGEN COMPOSITION White et al. 1257
osteogenesis impeifecta or Ehlers-Danlos.''^ '"^ Other strated they were not prone to hernia formation them-
prevtou.s work has also demonstrated reduced collageti selves, because 36 per cent had no abdominal surgical
I/Ill ratios in the .skin and fa.scia taken from the scar history before their enrollment in this study. However,
tissue of patients with incisional hernias and patients on subanalysis of only those control subjects with
with recurrent incisional hernias compared with that of prior abdominal surgical history and no hernia, our
heallhy tissue or scar from patients without her- results were not significantly altered.
nias.-- '^ Therefore, the understanding of the patho- Our findings suggest a possible pre-existing colla-
genesis of incisional and recurrent incisiona! hernia gen defect in the primary tissues of the abdominal wall
formation is based on the concept of abnormal colla- in patients with recurrent incisional hernias. We be-
gen metabolism either through impaired wound heal- lieve that further research using microarray technology
ing or impaired constitutive collagen expression and may elucidate a specific threshold or "protein signa-
formation associated with genetic disorders. ture" that could allow clinical screening for hernia
To our knowledge, this study is the first to examine formation risk. Such screening, if developed, could
nonscarred skin and fascial tissues of healthy control allow tailoring of future patients" surgical treatment so
subjects and compare them with nonscarred skin and as to minimize or avoid the risk of incisional hernia.
fascial tissues of patients with recurreni incisional her-
nias. This proved feasible by examining a patient Acknowledgments
population undergoing laparoscopic surgery, in which
We thank Kent van Sickle, M.D., for assistance in tissue
trocar incisions are typically made well away from the
collection and Dianne Lawson. Emory University Hospital
site of prior surgery or known pathology. This experi- Department of Pathology, for mounting and staining our
mental design allowed us to compare primary, non- biopsy specimens.
scarred tissue in both the experimental and control
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