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XI
42 Identification of the Patients at Risk
(for Recurrent Hernia Disease)
P.R. M, P. L-J, U. K
What Do We Know About the Pathogenesis
of Recurrent Hernia Disease?
Since its first description by Keith in the year 1924 on
the origin and nature of hernia (Keith A, Brit J Surg
11: 455–475), ample evidence has been collected that
the collagen composition is altered in wound-healing
disturbances that are summed up as recurrent hernia
disease. The predominant observation is that the type-I
to type-III collagen ratio is altered in such a way that
type-III collagen is more abundant in wound-healing
disturbances. Klinge et al. have confirmed this finding
in patients with inguinal and incisional hernia disease
[1, 2], and novel work indicates that such a disturbance
in collagen composition is also prevalent in patients
with hemorrhoids as well as diverticulosis [3]. Using
different protein and molecular biology techniques,
this finding was confirmed in tissue specimens from
patients who underwent surgical intervention for fail-
ure of mesh implantation [4]. The main question aris-
ing is which factor(s) direct the altered collagen ratio.
Undoubtedly, tissue fibroblasts are the major source
of interstitial collagens in tissue. Fibroblasts may not
be regarded as uniform cell type with uniform synthe-
sis profile. Interaction of fibroblasts with infiltrating
macrophages may have significant influence on their
expression profile [5]. Therefore, Klinge et al. set up
an in vitro model system with outgrowths of primary
fibroblasts from healthy controls and from patients with
recurrent hernia disease [6, 7]. This model system al-
lowed them to test whether the collagen synthesis rate is
different in fibroblasts obtained from these two groups
and circumvents the possible interference of infiltrat-
ing mononuclear cells. As major finding, Klinge et al.
determine that the fibroblasts in vitro exhibit the same
alterations of collagen synthesis as has been determined
in tissue specimen, indicating that a defect must reside
within the inherent fibroblast synthesis program.
How Is Wound Healing Coordinated?
Wound healing may be subdivided into different phases,
inflammatory, proliferative, and maturation [8]. The
dynamic nature of this process has to be kept in mind.
It is well known that exogenous factors like smoking
may affect wound-healing processes, e.g. by reducing
the blood flow due to its vasoconstrictive action [9] and
by interfering with the vitamin-C metabolism which
leads to a relative vitamin-C deficiency and diminished
collagen cross-linking [10]. Other exogenous factors are
comedications that are administered due to comorbidi-
ties like congestive heart failure or metabolic syndrome,
namely angiotensin-converting enzyme inhibitors and
statins. These have a well-proven impact on the activity
of matrix-degrading enzymes like matrix metallopro-
teinase-2 (MMP-2) and on the collagen synthesis rate it-
self [11]. However, the data outlined above indicate that
such confounding factors are not predominantly acting,
as fibroblasts in cell culture retain the same alterations
398 What Can we Do to Improve Our Results?
42
of the collagen synthesis rate or the factors must have a
long-lasting influence. Given epidemiological data and
the observation that a collagen disease may prevail in
some families, it is conceivable that a considerable effect
and influence must reside within the genes.
What Is Known About Collagen Genes
and Their Regulation?
The advent of molecular biology has revolutionized
our understanding of diseases. The number of genetic
diseases is still increasing as mutations within genes
that are causative are found. The most studied colla-
gen disease hitherto is the Ehler-Danlos syndrome in
which a mutation within the coding sequence of the
type collagen genes COL5A1 and COL3A1 are present
[12, 13]. Other known collagen diseases are the Marfan
syndrome, in which mutations of FBN1 on chromosome
15 or FBN2 on chromosome 5 are present [14] and the
osteogenesis imperfecta with mutations in the COL1A1
gene [15]. For recurrent hernia disease there is no single
mutation (e.g. single nucleotide polymorphism, SNP)
known from the literature that is causative for disease;
however, an in-depth understanding of the collagen
gene synthesis requires that the respective regulatory
elements relevant for gene transcription are known.
Such elements may be localized in immediate context
of the coding sequence, denoted as proximal promoter
sequences, interspersed in the gene within introns, or
they are localized in distance to the coding sequence,
up to 20 kb and more [16]. Regulatory elements may
act by enhancing or silencing gene transcription. It is
notable that the different components of collagens, e.g.
the alpha-1 and alpha-2 strand of the collagen type-I
trimer are regulated co-ordinately at the transcriptional
level [17]. Several groups have identified key regions
for the transcriptional regulation of type-I and type-III
collagen. These elements are bound by transcription
factors, amongst others activating protein 1 (AP1), Sp1,
Sp3, YB-1 and C/EBP [18–21], which are activated af-
ter cellular contact with cytokines like TNF-α, TGF-
β, IFN-α. For the understanding of gene regulation,
it is of paramount importance to keep in mind that
the programs for gene regulation are mostly set up in
a cell-specific fashion, meaning that fibroblasts may
orchestrate collagen synthesis completely differently
from macrophages, and knowledge from one cell type
may not be transferred to another cell type.
Hypothetically, there are two different ways of al-
tering the collagen gene regulation in a cell. The first
disorder relates to an altered signalling within a cell,
e.g. cytokine signalling transmitted to transcription fac-
tor activation. Such alterations of signalling may have
the same impact as gene mutations within regulatory
elements, which constitute the second possibility. Such
mutations within regulatory elements may prevent bind-
ing of transcription factors or enhance their binding
affinities. The complexity of the whole system cannot be
stressed enough, given that the transcription initiation
complex consists of at least 20 different proteins that
have to gather around the transcription start site.
Are There Candidate Genes for Wound
Healing Disturbances?
From other diseases with gradual and/or late onset in
life, like arterial hypertension or diabetes mellitus, it
might be anticipated that not a mono-causal genetic
defect exists but rather a whole array of genetic altera-
tions come together, making an individual susceptible
for a certain disease. However, knowledge about such
polygenetic traits has to be gathered before one may
assume such a defect. Regarding collagen gene regula-
tion, it is noteworthy that embryos with three copies
of chromosome 18 (trisomy 18) have only type-III col-
lagen in their skin, and lack type-I collagen [22]. Such
a profound alteration of collagen type-III synthesis
surprises, as the gene locus is located on chromosome
2q31 and the type-I collagen gene Col1A1 locus re-
sides on chromosome 17q21.33, the Col1A2 on 7q22.1.
One may postulate that there is an important genetic
factor located on chromosome 18 and that this factor
regulates the expression of the collagen genes inversely.
The identification of chromosome 18 as dominantly
acting on collagen expression renders all genes located
on this chromosome interesting as candidate genes for
wound-healing disturbances. Similarly, the collagen
genes themselves and aberrations within their promoter
sequences may be of relevance for wound-healing dis-
turbances. Recent results from our group indeed indi-
cate that such aberrations exist, that alter transcription
rates of collagen genes and that might be useful as pre-
dictive markers for wound-healing disturbances.
References
1. Klinge U, Zheng H, et al. (1999) Altered collagen synthesis in
fascia transversalis of patients with inguinal hernia. Hernia
4: 181–187
2. Klinge U, Si ZY, et al. (2000) Abnormal collagen I to III distribu-
tion in the skin of patients with incisional hernia. Eur Surg
Res 32(1): 43–48
399
XI
Identification of the Patients at Risk (for Recurrent Hernia Disease)
3. Stumpf M, Cao W, et al. (2001) Increased distribution of col-
lagen type III and reduced expression of matrix metallopro-
teinase 1 in patients with diverticular disease. Int J Colorectal
Dis 16(5): 271–275
4.
Junge K, Klinge U, et al. (2004) Decreased collagen type I/III
ratio in patients with recurring hernia after implantation of al-
loplastic prostheses. Langenbecks Arch Surg 389(1): 17–22
5. Wynes MW, Frankel SK, et al. (2004) IL-4-induced macro-
phage-derived IGF-I protects myofibroblasts from apopto-
sis following growth factor withdrawal. J Leukoc Biol 76(5):
1019–1027
6. Si Z, Bhardwaj R, et al. (2002) Impaired balance of type I and
type III procollagen mRNA in cultured fibroblasts of patients
with incisional hernia. Surgery 131(3): 324–331
7. Rosch R, Junge K, et al. (2003) Hernia a collagen disease? Eur
Surg Res 35(1): 11 15
8. Waldorf H, Fewkes J (1995) Wound healing. Adv Dermatol
10: 77–96; discussion 97
9. Silverstein P (1992) Smoking and wound healing. Am J Med
93(1A): 22S–24S
10. Yin L, Morita A, et al. (2000) Alterations of extracellular ma-
trix induced by tobacco smoke extract. Arch Dermatol Res
292(4): 188–194
11. Jesmin S, Sakuma I, et al. (2003) Role of angiotensin II in
altered expression of molecules responsible for coronary
matrix remodeling in insulin-resistant diabetic rats. Arte-
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12. Zoppi N, Gardella R, et al. (2004) Human fibroblasts with mu-
tations in COL5A1 and COL3A1 genes do not organize colla-
gens and fibronectin in the extracellular matrix, down-regu-
late alpha2beta1 integrin, and recruit alphavbeta3 Instead of
alpha5beta1 integrin. J Biol Chem 279(18): 18157–18168
13. Malfait F, De Paepe A (2005) Molecular genetics in classic
Ehlers-Danlos syndrome. Am J Med Genet C Semin Med
Genet 139(1): 17–23
14. Rokicka A, Rok icki W, et al. (2002) [Genetic aspects of Marfan
syndrome]. Wiad Lek 55(1 2): 107–111
15. Qin W, He JX, et al. (2005) [Mutation detection of COL1A1
gene in a pedigree with osteogenesis imperfecta]. Yi Chuan
Xue Bao 32(3): 248–252
16. Mautner J, Behrends U, et al. (1996) c-myc expression is ac-
tivated by the immunoglobulin kappa-enhancers from a
distance of at least 30 kb but not by elements located within
50 kb of the unaltered c-myc locus in vivo. Oncogene 12(6):
1299–1307
17. Norman JT, Lindahl GE, et al. (2001) The Y-box binding pro-
tein YB-1 suppresses collagen alpha 1(I) gene transcription
via an evolutionarily conserved regulatory element in the
proximal promoter. J Biol Chem 276(32): 29880–29890
18. Mertens PR, Harendza S, et al. (1997) Glomerular mesangial
cell-specific transactivation of matrix metalloproteinase
2 transcription is mediated by YB-1. J Biol Chem 272(36):
22905–22912
19. Chen SJ, Artlett CM, et al. (1998) Modulation of human
alpha1(I) procollagen gene activity by interaction with
Sp1 and Sp3 transcription factors in vitro. Gene 215(1):
101–110
20. Dhalla AK, Ririe SS, et al. (1998) chk-YB-1b, a Y-box binding
protein activates transcription from rat alpha1(I) procollagen
gene promoter. Biochem J 336: 373–379
21. Mertens PR, Steinmann K, et al. (2002) Combinatorial inter-
actions of p53, activating protein-2, and YB-1 with a single
enhancer element regulate gelatinase A expression in neo-
plastic cells. J Biol Chem 277(28): 24875–24882
22. Brand-Saberi B, Epperlein HH, et al. (1994) Distribution of
extracellular matrix components in nuchal skin from fetuses
carrying trisomy 18 and trisomy 21. Cell Tissue Res 277(3):
465–475
Discussion
Kehlet:
I think you have opened up a new field in hernia
surgery. Very important. If you can use this test, what
will it cost?
Mertens:
We are at a very early stage. We have to test
this with a larger cohort of patients but I think it is very
promising. When you do it like that you are in a range
of 40–50 €. That’s it.
Franz: Maybe I missed a slide. Where do the fibroblasts
from the control patients come from? And where do the
fibroblasts from the hernia patients come from?
Mertens:
This is DNA extracted from blood. It is not
DNA extracted from fibroblasts. Actually, this is a very
interesting question we are addressing right now. Because
when you are a mosaic, that means not everywhere in
the body that microsatellite has to be the same. Maybe
on the head it is different compared to the feet. At the
side of your hernia where you have damaged your tissue
it might be different. One of the aspects which is very
interesting is that you told me smoking has a bad impact
on recurrence rate and you do not lose that even if you
are a long-term non-smoker. So maybe smoking affects a
lot of things, like folat metabolism, like vitamin C meta-
bolism and what we are just trying to address is whether
this microsatellite instability is brought forward by these
deficiencies so that you keep this trait, this genetic trait,
lifelong, which would make sense. The other thing is age-
dependent changes. You do not have the same incidence
of recurrences at every age. So there must be something
that changes with age.
XI
43 The Biological Treatment
of the Hernia Disease
M.G. F
The biological treatment of hernias is perhaps best de-
fined as a bio-molecular or biomechanical therapy that
results in the complete and sustained correction of the
structure and function of a hernia defect. Biological
therapy of hernias could be applied as an adjuvant to
surgical reconstruction, or, potentially stand alone as
a primary therapy.
Growing evidence suggests that recurrent hernias
are most often the result of surgical wound failure. Our
group approaches the problem of incisional hernias
with the hypothesis that the majority of incisional her-
nias develop following the mechanical disruption of
early laparotomy wounds. By extension, most recur-
rent hernias are the result of mechanical wound failure
during the lag phase of the wound healing trajectory
(
⊡
Fig. 43.1). Traditional surgical teaching is that lapa-
rotomy wound failure is a rare event, with reported
dehiscence rates clustered around 0.1% [1–3]. The
parallel wound-healing literature therefore suggested
that incisional hernias were the result of late laparotomy
wound failure and scar breakdown [4] This concept was
first challenged by clinical studies of incisional hernias
that recorded high primary and secondary recurrence
rates after short-term follow-up, typically 2–4 years [5,
time
platelets
neurophils
wound strength
collagen
macrophages
broblasts
lag phase
capillaries and epithelium
lymphocytes
specic cell, matrix or process %healing %
⊡
Fig. 43.1. The cellular and molecular
pathways of surgical wound repair are
time-dependent. During the initial lag-
phase, wound tensile strength is low
402 What Can we Do to Improve Our Results?
43
6]. One prospective study found that the true rate of
laparotomy wound failure is closer to 11%, and that the
majority of these (94%) go on to form incisional hernias
during the first 3 years after abdominal operations [2]
(
⊡
Table 43.1). The real laparotomy wound failure rate
is therefore 100 times what most surgeons think it is.
In simplest terms, most incisional hernias and recur-
rent hernias are therefore derived from clinically occult
dehiscences. The overlying skin wound heals, conceal-
ing the underlying myofascial defect. This mechanism
of early mechanical laparotomy wound failure is more
consistent with modern acute wound healing science.
There are no other models of acute wound healing sug-
gesting that a successfully healed acute wound goes on
to breakdown and mechanical fail at a later date. This
mechanism is also unique in that it assumes that the
majority of abdominal wall laparotomy wound failures
occur in hosts with no clearly identifiable wound-heal-
ing defect. The majority of surgical patients undergoing
laparotomy are considered safe surgical candidates from
a wound-healing perspective.
The time-course of recurrent hernia formation is,
however, debated [7, 8]. Incisional hernias detected
many years after an operation have been ascribed to
impaired collagen and tissue protease metabolism [9,
10]. Incisional herniation was associated with increased
collagen solubility, increased ratios of early wound ma-
trix collagen isoforms (e.g. collagen III) and increased
metalloprotease 9 levels (MMP-9) [11, 13]. These stud-
ies were limited by the small numbers of patients and no
confirmatory animal model data exist. A decreased ratio
of type I: type III collagens was detected in fascial and
skin specimens obtained from patients with incisional
hernia disease at both the mRNA and the protein level
[9, 14]. Morphological changes were present not only in
the fascial tissue, but also in the hernia sac, skin speci-
mens and scar tissue surrounding explanted meshes
of hernia patients; collectively, the changes indicate a
generalized alteration of collagen metabolism. MMPs
are required for the proper progression and maturation
of wound healing. Whereas MMP-2, -7, -8, and -9 are
absent in healthy skin, MMP-1, -2, -3, -9, -11, -13, -14
are upregulated after injury. MMP-2 overexpression
has been measured in fibroblasts of patients with direct
inguinal hernia formation and MMP-13 overexpression
was detected in specimens obtained from patients with
recurrent inguinal hernias. In most cases, recurrent
inguinal hernias most likely are a form of incisional
hernia. Interestingly, there is a link between MMP-2
and collagen gene expression, as collagen binding to
discoidin domain receptor II (DDR 2) regulates MMP-2
gene transcription [15]. Correspondingly, alterations in
both MMP-2 and fibrillar collagen can interfere with
the wound-healing process. Studies hypothesizing that
hernia formation is a disease of the extracellular matrix
(ECM) are a new approach to the biology of recurrent
inguinal hernia and incisional hernia formation.
Surgical approaches to hernias are primarily based
on mechanical principles. Fundamental techniques
involve the sturdy apposition of durable aponeurotic
tissues like the transversalis fascia, conjoined tendon,
inguinal ligament, Cooper’s ligament or rectus sheath.
Consistently high recurrence rates led to the concept
of a tension-free repair and the introduction of allo-
plastic mesh implants [16, 17]. The first suggestion of
a biological approach soon followed, when it was hy-
pothesized that part of the mechanism for the reduced
recurrence rates observed with mesh implants was the
avoidance or replacement of diseased or structurally
defective tissue in hernia repairs. The current wide-
spread use of plastic mesh implants has not, however,
solved the problem of hernia recurrence. The problem
is especially evident with recurrent incisional hernias,
where population-based and administrative database
analyses find a consistently high recurrence rate of be-
tween 24 and 58% and the more recent conclusion that
the mesh repair of incisional hernias does not reduce
the incidence of recurrence, but only increases the time-
interval until the recurrence is recognized [18].
We hypothesize that the natural delay in acute
wound healing prevents the establishment of adequate
tensile strength within laparotomy wounds to off-set the
loads placed across it by recovering surgical patients.
An improved understanding of the cellular and mo-
lecular mechanisms of acute tissue repair now makes a
biological approach to the problem of incisional hernia
formation possible. Fundamentally, an incisional hernia
occurs as the result of the biomechanical failure of an
acute laparotomy wound. It is during the earliest pe-
riod of acute wound healing when the wound depends
⊡
Table 43.1. Human laparotomy wound-edge gap on
post-operative day 30
Laparotomy wound
outcome at 43 months
Less than
12 mm
More than
12 mm
Healed [%] 95
(140/147)
6
(1/18)
Incisional hernia [%] 5
(7/140)
94
(17/18)
403
XI
The Biological Treatment of the Hernia Disease
entirely on suture integrity to maintain abdominal wall
closure. Recurrences following mesh repairs occur al-
most exclusively at the suture line interface between
the implant material and the laparotomy wound. Nu-
merous studies conclude that the breaking strength of
mesh implants themselves far exceeds wound-breaking
strength, and that, in fact, most of the mesh implants in
clinical use are over-engineered [19]. It is an unusual
event for a permanent mesh implant to mechanically
fail. Most hernia repair patients are recovering from
their procedures and returning to increased levels of
activity and placing increasing loads across the acute
wound during its weakest phase.
The potential for biomolecular intervention for
surgical problems has improved with the continued
maturation of applied biomolecular engineering and
the wealth of data generated by the various genome
projects. It is no longer impossible to imagine impor-
tant applications of biotherapy in the field of wound
healing (benign neoplasia or regenerative medicine)
for unsolved problems like surgical wound failure (re-
current hernias), hypertrophic scar, bony non-union,
gastrointestinal anastamoses, vascular endoleaks and
intravascular stent patency.
Growth factors were one of the first groups of bio-
active molecules identified for clinical application. De-
rived primarily from tissue macrophages and wound-
repair fibroblasts, tissue growth factors are an important
class of tissue repair, signaling peptides upregulated
during the lag phase of acute wound healing [20, 21].
Five to seven days are required, however, before peak
levels of fibroproliferative growth factors like transform-
ing growth factor beta (TGF-β) are reached within acute
wounds. Acute wound therapy with proliferative growth
factors is known to accelerate the appearance of fibro-
blasts and collagen into the wound thereby shortening
the natural lag phase for gain in injured tissue tensile
strength. Several reports have demonstrated the ability
of platelet-derived growth factor (PDGF) and TGF-β to
accelerate the recovery of tensile strength in acute skin
and laparotomy incisions [22–24].
A series of time-dependent cellular and molecular
events must be activated and orchestrated during the
establishment of a surgical wound matrix (
⊡
Fig. 43.1).
One strategy for the biological treatment of recurrent
hernias would be to accelerate tissue repair, or shift
the temporal wound-healing trajectory to the left
(
⊡
Fig. 43.2
). It was already noted, that in addition to
the natural delay in the activation of tissue repair, ab-
normal wound matrix structure may contribute to the
mechanism of recurrent hernias. Biological therapies
might also be designed to correct abnormal collagen
isoform expression, or block overexpressed MMP ac-
tivity. Ideally, normal aponeurotic, fascial, muscle or
tendon structure would be regenerated following hernia
repairs. Biological approaches for normal laparotomy
wounds might be guided by information gained from
identified genetic or epigenetic pathways associated
with hernia formation like abnormal collagen matrix
structure in the Ehlers-Danlos syndrome or MMP/
TIMP expression in abdominal aortic aneurysm (AAA)
disease or in other chronic wounds.
Exogenous application of cytokine growth factors
has been used to accelerate healing of chronic wounds.
Steed et al. first reported a successful clinical trial using
recombinant platelet-derived growth factors (PDGF-BB)
to accelerate healing of diabetic foot ulcers [25]. PDGF
has also been reported to shorten the time of healing for
⊡
Fig. 43.2. Accelerating the pathways of
surgical wound healing can be visualized
as a shift-to-the-left of the wound-healing
trajectory. We call this surgical priming
time
wound
breaking strength
surgical wound priming
404 What Can we Do to Improve Our Results?
43
pressure ulcers. Robson et al. demonstrated that basic
fibroblast growth factor (bFGF) could decrease the size
of pressure ulcers more effectively than a placebo control
[26]. Keratinocyte growth factor-2 (KGF-2) appears to
have been effective in achieving closure in venous stasis
ulcers, as has transforming growth factor beta-2 (TGF-
β2). Bone morphogenetic proteins (BMP) are in the
family of fibroblast growth factors and are now approved
by the United States Food and Drug Administration
(FDA) for the acceleration of interbody fusion following
anterior diskectomy during spine operations [27].
Success in treating chronic wounds with exog-
enous cytokine growth factors has been disappoint-
ing. Despite numerous clinical trials, only one growth
factor has been approved by the FDA for clinical use.
There are several reasons for this. The chronic wound
has a significant bacterial burden which is often not
controlled prior to treatment with growth factors.
Since bacteria degrade cytokine growth factors, larger
amounts are required to be effective. In addition to
the degradation of the cytokine growth factors, the
bacteria produce proteases and MMPs both by them-
selves and as a product of bacterial action on tissue
cells. These proteases and MMPs can degrade not only
the growth factor molecules but also the cytokine re-
ceptors.
The acute wound, especially the surgical incision, is
a closed wound with a bacterial burden 100–1000-fold
less than the chronic wound [28]. The surgical wound
may therefore be the ideal application for the tissue
growth factors shown to accelerate the rate of gain of
tensile strength in experimental incisions (
⊡
Fig. 43.2
).
Preliminary studies showed that not only can growth-
factor therapy be done at the time of surgical incision,
it can be effective when initiating the inflammatory
phase of healing prior to tissue injury by injecting cyto-
kine growth factors in the area of the intended incision
[23, 29]. Combining this pretreatment with a second
growth-factor application at the time of incision nearly
doubled the breaking strength of the acute wound
(Franz, personal communication, date??).
Botsford first introduced the concept of accelerat-
ing surgical wound healing when describing delayed
primary closures. It was observed that surgical wounds
closed 7 days after incision regained tensile strength
faster than incisions closed primarily [30]. The im-
proved understanding of the cellular and molecular
mechanism of surgical wound healing led to the pro-
posal that it is the recruitment of repair cells and mol-
ecules into the open surgical incision that is the mecha-
nism for accelerated repair following delayed primary
closure. We call this phenomenon surgical priming. The
availability of cloned biological signalling molecules
now makes it possible to biopharmacologically induce
the appearance of tissue repair cells, avoiding the practi-
cal difficulties associated with the surgical technique of
delayed primary closure. The technique of delivering
tissue repair growth factors at the time of incision is
called simple priming and the technique of delivering
the same growth factors several days prior to incision
is called “early priming”.
Growth factor strategies in hernia repairs:
▬ Accelerate recovery of wound tensile strength
▬ Optimize wound collagen structure and function
▬ Stabilize implant incorporation
Materials and Methods
We developed a model of incisional hernias in order
to study the mechanism of acute wound healing dur-
ing hernia formation and recurrence. We based our
animal model of laparotomy wound failure and in-
cisional hernia formation on the best available clinical
evidence. Well-controlled, prospective studies conclude
that most laparotomy wound disruptions progressing to
incisional hernias begin to form soon after laparotomy
wound closure [2, 31]. In the model, laparotomy wounds
on the ventral abdominal wall of rats are temporarily
repaired with rapidly absorbed plain 6–0 catgut suture.
Laparotomy wound-edge separation occurs, progress-
ing to incisional hernia formation due to incompletely
supported mechanical loads. The incisional hernias that
develop have well-defined hernia rings, hernia sacs and
⊡
Fig. 43.3. In our animal models, mechanical failure of the
laparotomy wound occurs and progresses to incisional hernias.
The gross and microscopic structure is very similar to the hu-
man defect. In the rat, the skin flap serves as a sling to support
the viscera for experiments requiring early disruption of the
laparotomy wound
405
XI
The Biological Treatment of the Hernia Disease
visceral adhesions, all characteristic of the incisional
hernias that develop in humans (
⊡
Fig. 43.3
). In one
variant of this model, the laparotomy incision is inten-
tionally left open. These animals go on to form chronic,
giant, incisional hernias [32]. We feel our model is an
improvement over those that use abdominal wall exci-
sions with inherent loss of abdominal wall muscular and
tendinous structure. That approach does not control
for the function of intact anatomical structures during
laparotomy repair, including distractive load forces gen-
erated by the lateral oblique and midline rectus muscle
and fascia components.
Aqueous Priming with TGF-beta2
Under our experimental protocol, 100 l of vehicle
(sodium acetate aqueous solution) or 100 l of vehi-
cle containing recombinant human TGF-β2 (1 gm)
(Genzyme, Framingham, MA) was injected evenly into
the linea alba using a tuberculin syringe immediately
prior to fascial incision (simple priming,
⊡
Fig. 43.4).
A 5.0-cm midline celiotomy was then performed and
the laparotomy incision was immediately closed with
a single, 5–0 plain catgut suture placed across the mid-
wound, as described above.
All rats were sacrificed on postoperative day (POD)
28 by intraperitoneal Nembutal overdose (100 mg/kg).
The entire ventral abdominal wall was then excised and
the skin was separated from the musculo-fascial layer.
The peritoneal and subcutaneous surfaces of each ab-
dominal wall were then carefully inspected for the pres-
ence of herniated abdominal contents. A minimum of
2 mm fascial separation was required for a wound to
be scored as containing an incisional hernia. Sagittal
fascial wound and/or hernia sections were then cut and
immediately fixed in 10% neutral buffered formalin in
preparation for histological analysis.
Continuous Release Therapy with Basic
Fibroblast Growth Factor
The first set of experiments was designed to test the
activity of bFGF in preventing the development of inci-
sional hernias. In the hernia prevention group (n = 20),
a pliable, delayed-release polygalactone polymer-rod
formulation of basic fibroblast growth factor (bFGF-rod;
10 µg, Scios Inc, Sunnyvale, CA) was sewn to the ventral
edge the primary fascial wound closure (
⊡
Fig. 43.5).
Each rod was 5 cm in length and 1 mm in diameter.
Previous experience in our laboratory established 10 µg
as the optimal dose to stimulate the angiogenic and
fibroplastic properties of bFGF in vivo. A growth fac-
tor delivery control group (n = 20) had rods without
bFGF incorporated into the fascial closure (empty rod
control). In the hernia model control group (n = 30), no
rods were placed (hernia model control). The incidence
of primary incisional hernia was determined during
necropsies performed on postoperative day (POD) 28
on ten animals each in the bFGF rod treatment and
empty rod control groups.
The recovery of fascial wound mechanical strength
was measured on POD 7 using ten rats each from the
bFGF-rod treatment and empty-rod control groups.
peritoneal injection of Nembutal (100 mg/kg) and the
entire ventral abdominal wall was excised. Following
separation of the skin, the musculo-fascial layer was
divided into three 1-cm-wide by 4-cm-long strips cut
⊡
Fig. 43.4. During biological priming, a potential surgical
wound site is treated with fibroproliferative and angiogenic
tissue repair growth factors before the incision is made
⊡
Fig. 43.5. A delayed-release
polymer containing tissue re-
pair growth factors can be
incorporated into a surgical
wound during hernia repairs
406 What Can we Do to Improve Our Results?
43
perpendicular to the fascial wound for biomechanical
analysis. Breaking strength analysis was performed on
the fascial wound strips using an Instron pneumatic
tensiometer (Model MN44, Instron Corporation, Can-
ton MA) set at a crosshead speed of 10 mm/min. The
breaking strength was defined as the force in Newton’s
required to rupture the resultant incisional scar. Blood
samples were obtained at necropsy and centrifuged for
ELISA immunoassay analysis of serum bFGF levels
(R&D Systems Inc. Minneapolis, MN).
The second set of experiments was designed to
test whether bFGF augments the acute fascial repair
of mature incisional hernias (recurrent hernia experi-
ment). Twenty seven incisional hernias developed in
the hernia model control group of 30 rats. The hernias
were reduced and fascial defects repaired. The rats
were then randomized to receive either bFGF rod her-
nia treatment (n = 13) or control empty-rod therapy
(n = 14). The incidence of recurrent incision hernias
was determined during necropsies performed 28 days
after hernia repair. Several 2-mm biopsies taken of the
fascial: fascial interface or hernia ring were snap frozen
in liquid nitrogen for gene expression analysis. Inflam-
matory cell infiltrate and wound angiogenesis were
measured using hematoxylin and eosin and trichrome
staining. The intensity of wound collagen type-1 deposi-
tion was measured by quantifying the digital intensity
of staining over three HPF using computer-aided image
analysis software (Scion Image, Scion Corp., Bethesda,
MD).
Statistical Analysis
Data were analyzed using SigmaStat software (Jandel
Scientific, Corte Madera, CA). The Fisher’s exact test
was used to determine differences in the incidence of
incisional hernias. Student’s t test or ANOVA was used
to determine differences in cell count, collagen stain-
ing, angiogenesis and collagen mRNA band intensity
following RT-PCR. P values of < 0.05 were considered
significant.
Results
TGF-beta 2
Incisional hernias developed in 88% (35/40) and 79%
(11/14) of the untreated or vehicle-treated fascial in-
cisions, respectively, after 28 days. No incisional her-
nias (0/16) occurred in the TGF-β2 treated wounds
(
⊡
Table 43. 2
; p < 0.05). All animals gained weight dur-
ing the study period and there was no difference in the
mean weight between all three groups after 28 days
(group 1 306 +/– 11.7 g vs. group 2 312 +/– 14.1 g vs.
group 3 311 +/– 12.7 g). There were no deaths during
the study period and no evidence of evisceration and
or intestinal incarceration, obstruction or strangul-
ation.
Cellular infiltration into the wounds on day 28
consisted mainly of macrophages, lymphocytes and
fibroblasts. Fibroblast number after 28 days was
higher within the TGF-β2-treated fascial incisions
(p < 0.05). Trichrome and collagen immunostain-
ing also was increased in fascial wounds treated with
TGF-β2.
bFGF Delivery
Sustained delivery of bFGF was documented 7 days
following surgical implantation. Analysis of POD
7 serum revealed a significant increase in systemic
bFGF delivery compared to controls (0.033 µ + 0.017
bFGF rod treatment, 0.012 µ + 0.0097 empty rod
control, and 0.010 µ + 0.008 hernia model control,
p < 0.05)
Breaking Strength
All wounds mechanically disrupted at the fascial:fas-
cial interface. Wounds treated with bFGF impregnated
rods developed increased breaking strength in the early
postoperative fascial incision. The bFGF rod-treated
animals breaking strength was 12.29 N +/– 2.98 vs.
8.61 N +/– 3.17 in the empty-rod control animals’
(p < 0.05).
⊡
Table 43.2. Growth factor priming (TGF)
Treatment modality No. Incisional
hernias [%]
Untreated 40 35 (88)
Vehicle prophylaxis 14 11 (79)
TGF-β2 prophylaxis 16 10 (0)
a
a
Significantly less than untreated or vehicle prophylaxis.
407
XI
The Biological Treatment of the Hernia Disease
Prevention of Incisional Hernia Development
with bFGF
Incisional hernia formation was significantly reduced
with bFGF rod therapy. Incisional hernias devel-
oped in 90% (27/30) of control incisions compared
to 60% (6/10) in empty rod control-treated incisions,
and only 30% (3/10) of the bFGF impregnated rod-
treated incisions after 28 days (p < 0.05,
⊡
Table 43.4).
Full-thickness fascial sectioning perpendicular to the
wound revealed the polymer rod incorporated into
the provisional matrix. There were no deaths dur-
ing the study period and no evidence of eviscera-
tion, intestinal incarceration, obstruction or stran-
gulation. There were no obvious systemic effects of
the bFGF observed in the postoperative period or at
necropsy.
Treatment of Mature Incisional Hernias
with bFGF
Incisional hernia recurrence was significantly reduced
with bFGF rod therapy. The 27 incisional hernias that
developed in the hernia model control group were op-
eratively repaired 28 days after the initial procedure.
Prior to repair, the rats were randomized to receive
the 10 g bFGF polymer or placebo polymer incor-
porated into the fascial closure. After another 28 days,
86% (12/14) of the empty rod control-treated hernia
repairs developed recurrent hernias compared to
only 23% (3/13) of the bFGF treated group (p < 0.05)
(
⊡
Table 43. 3).
Inflammatory Infiltrate
Basic histology demonstrated that rods were still pres-
ent, although approximately 50% smaller than when
initially applied to the wound, demonstrating a slow
absorption pattern of the polygalactone rod polymer.
There was no difference in size between the bFGF-rod
and empty-rod absorption or incorporation into the
wound. There was a modest non-granulomatous in-
flammatory infiltrate surrounding the bFGF and empty-
rod polymers. H&E staining suggested a gradient effect
for polymorphonuclear leukocytes and macrophages in
the bFGF-treated incisions.
Angiogenesis
Grossly, there was a visible increase in granulation tissue
deposition adjacent to the bFGF rods when compared
to the control animals. Dense capillary and provisional
matrix deposition was observed in bFGF-treated in-
cisions, resulting in macroscopic nodules similar to
small hemangiomas. Microscopic neovascularization
was most pronounced adjacent to the bFGF rods, and
decreased as a function of distance from the rod.
Collagen Production
Basic FGF had no significant effect on wound collagen
mRNA levels by POD 28. Histology and immunohis-
tochemistry, however, demonstrated a gradient effect
for fibroblast chemotaxis and collagen protein produc-
tion in bFGF-treated incisions. Collagen I staining was
significantly increased adjacent to the polymer by POD
7 (103.72 µ + 1.70 vs. 24.42 µ + 6.56, p < 0.01). This
intense collagen staining decreased as a function of
distance from the rod.
Discussion
New approaches to the problem of abdominal wall
wound failure are needed because the 11% incidence
of primary and 24–58% incidence of recurrent inci-
sional hernias remain unchanged over 50 years [5,
18, 33]. Mechanical approaches alone including op-
timized suture length to wound length ratios, inci-
sion location and orientation, layered versus mass
closure and even mesh herniorrhaphy have all failed
to significantly impact this very common surgical
complication. Mesh herniorrhaphy is the only surgical
⊡
Table 43.3. Growth factor priming (bFGF)
Treatment modality No. Incisional
hernias [%]
Untreated 40 35 (88)
Placebo prophylaxis 10 16 (60)
bFGF prophylaxis 10 13
a
(30)
Placebo therapy 14 12 (86)
bFGF therapy 13 13
b
(23)
a
for p < 0.05 compared to untreated;
b
for p < 0.05 com-
pared to placebo therapy (Fisher’s exact test)
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