1 Women and Infants Hospital, Providence, RI 02905 and 2 Confluent Surgical Inc., 101A First Avenue, Waltham, MA 02451, USA
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Abstract |
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Key words: adhesion prevention/post-surgical adhesions/large animal models/laparoscopy/adhesion barriers
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Introduction |
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Post-operative adhesions are a common cause of small bowel obstruction and re-operation for pelvic pain, and adhesions involving the ovaries or Fallopian tubes are responsible for 1520% of female infertility cases (Ray et al., 1998). In addition to these adverse clinical sequelae, the economic impact in the USA in 1994 was significant with a direct cost of $1.33 billion for all hospitalizations during which adhesiolysis was performed, based on Ray's analysis (Ray et al., 1998
). Other studies have shown that of surgical patients, 35% were readmitted at least once for problems directly or possibly related to adhesions over a 10 year period (Ellis et al., 1999
; Lower et al., 2000
).
Although the ultimate solution to this problem will probably result from an increased understanding of the humoral agents and cellular events that control adhesion formation, current clinical needs could be met by an effective adhesion barrier that is easy to apply in both open and laparoscopic procedures.
The majority of models developed to study adhesion barriers utilize small animals, such as rats (Golan et al., 1995; Hellebrekers et al., 2000
), mice (Haney and Doty, 1992
) and rabbits (Marana et al., 1997
). These models use a variety of means to mimic surgical injury, such as abrasion or electrocautery applied to a range of organs including uterine horns (Golan et al., 1995
), the caecum, ovaries (Marana et al., 1997
) and the pelvic sidewall to create a nidus for adhesion formation. These models have contributed greatly to our knowledge of adhesion formation and prevention. However, due to differences in scale between humans and these models, they are limited in their ability to predict clinical success.
Two studies have previously used porcine adhesion models (Montz et al., 1993a; Christoforoni et al., 1996
), and in this study a porcine adhesion model has been developed with the intent to better mimic conditions involved in pelvic surgery. This model involves a clinically relevant surgical injury, in a species with organ size and weights similar to man. This model was used to evaluate the efficacy of a new synthetic absorbable polyethylene glycol product, SprayGelTM Adhesion Barrier (Confluent Surgical Inc., Waltham, MA, USA).
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Materials and methods |
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Animal Preparation and Operative Procedure
10 female virgin hogs weighing 5075 lbs were received and acclimatized for a minimum of 2 days prior to surgery. They were induced with a combination of tiletamine-zolazepam (5 mg/kg), xylazine (2 mg/kg) and atropine (0.05 mg/kg) administered i.m. Pre-operatively, the subjects received 1 gm cefazolin i.v. Following induction of the general anaesthetic the animals were maintained on a mix of isoflurane and oxygen inhalation for anaesthesia for the duration of the procedure. The abdominal region was shaved, scrubbed and draped in preparation for sterile surgery.
The celiotomy was created via a single, midline abdominal incision from the umbilicus to the symphysis pubis. The subcutaneous tissue and fascia were divided using electrocautery (Valley Lab Force 2, 35 watts cutting current; Valley Labs, Boulder, CO, USA). The subjects were placed in a Trendelenberg position, and the bladder was aspirated by cystostomy with electrocautery and wall suction or 18-gauge needle. Dry surgical gauze, towels and retractors were used to obtain adequate exposure to the pelvic side wall during the injury process. Both uterine horns were sharply transected at their midpoint after coagulation with monopolar electrocautery (25 watts coagulating current), and the transected ends of each were then re-anastomosed (end-to-end) using two interrupted sutures (30 Vicryl; Ethicon, Sommerville, NJ, USA). The parietal peritoneum of the pelvic side wall opposed to each uterine horn was then sharply excised from the analogue of the round ligament to the infundibulopelvic ligament to expose an area about 5x4 cm on the pelvic sidewall. Monopolar electrocautery (25 watts coagulating current) was used to obtain haemostasis where needed. One subject required suture ligation of a large bleeder of the pelvic sidewall. Following bilateral peritoneal excision and uterine horn anastomoses, both sidewalls and uterine horns were irrigated with saline to ensure adequate haemostasis and reduce tissue drying. Figure 1 shows an intraoperative view of a completed peritoneal injury, just prior to uterine horn transection and anastomosis.
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Application of adhesion barrier
The SprayGel Adhesion Barrier (Confluent Surgical) consists of two synthetic liquid precursors that, when mixed together, rapidly cross-link to form a solid, absorbable biocompatible hydrogel in situ. No external energy sources are required for polymerization, which is substantially completed within a few seconds with no heat evolution.
Both precursor solutions contain upwards of 90% water. The first precursor solution contains a modified polyethylene glycol (PEG) polymer with terminal electrophilic ester groups while the other precursor solution contains PEG that has nucleophilic amine end-groups. This second precursor solution also contains methylene blue, a colourant that is added to the formulation to facilitate visualization of the hydrogel. The SprayGel barrier is formulated to remain adherent to the site of application for approximately five days. At approximately that time the barrier breaks down by the process of hydrolysis, and the liberated water-soluble PEG molecules (<20 KDa) are absorbed and undergo renal clearance (Yamaoka et al., 1993). PEG molecules of this size have been shown to have a clearance half-life of about 15 min in mice (Yamaoka et al., 1993
).
SprayGel has passed a complete battery of tests including cytotoxicity, genotoxicity, haemolytic potential, sensitization and irritation. It does not affect wound healing or potentiate infections, and has been shown to be non-toxic at 30 times the expected human dose.
After the completion of injuries and randomization, the surgeon applied the adhesion barrier only to the pelvic side wall injury site assigned to the treatment group. No material was applied to the uterine horn. Figure 2 shows a typical application at a treated site. An air-assisted sprayer (Confluent Surgical), shown in Figure 3
, was used to carry out the deposition. The barrier was applied to achieve a thickness so that fine tissue structures under the barrier such as small blood vessels or muscle fibres became difficult to visualize due to the methylene blue colourant in the gel. This was previously established to be a thickness of 12 mm. The barrier was applied to the exposed subperitoneal muscle, and extended beyond the cut border by 23 cm to ensure coverage of potential ischaemic areas. Approximately 5 ml of each precursor was needed to cover an area of 12x8 cm. Due to the hydrophilic nature of the hydrogel barrier, treated sidewalls were irrigated with normal saline to ensure a moist, lubricious surface prior to closure.
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Following adhesion scoring, animals were euthanized with an i.v. potassium chloride injection, and a midline laparotomy was performed. From this approach the laparoscopically obtained adhesion scores could be confirmed, and representative tissues were retrieved for histological examination. Tissues were fixed in neutral buffered formalin, embedded, sectioned and stained using haematoxylin and eosin for light microscopy.
Statistics
To determine the statistical significance of adhesion formation incidence between the treated and control groups, a 2 test was used. Differences in the extent and severity of adhesion formation scores were assessed using the Wilcoxon signed rank test for nonparametric data. All data were analyzed using the SPSS software package (SPSS Version 9.0; SPSS, Chicago, IL, USA). A value of P < 0.05 was considered to be statistically significant.
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Results |
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One animal had no adhesions to either the control or treatment sites. Three animals had adhesions to both the control and treatment sites. No animals had adhesions to only the treatment site. Three of the 10 sites randomized to adhesion barrier treatment were involved in adhesions. One of these treated sites with adhesions had required suture ligation of a large arterial bleeder on the side wall during the initial surgery that could not be controlled with electrocautery alone prior to randomization to treatment. The remaining 7 of 10 treated sites were adhesion free.
When one compares the incidence of adhesion formation in the treated and control sites, a statistically significant reduction of 67% was observed in the treated sites (P = 0.006). Also, a statistically significant reduction in the adhesion extent score (P = 0.029) and adhesion severity score (P = 0.023) was observed in the treated sites. It is notable that when evaluated without the first two (6 day) cases the differences between incidence, extent and severity are no longer significant, probably because of the reduced sample size (n = 8). For representative purposes, Figure 4A shows a gross evaluation of a treatment site without adhesions, while Figure 4B
shows a control site with adhesions.
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Discussion |
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For a barrier to be clinically effective, it should be easy to use in both laparoscopic and open procedures, as well as adhere to the desired tissue long enough to prevent adhesions. Small animal models for adhesion barriers typically do not allow laparoscopic device application, and organ sizes and forces are insufficient to challenge the ability of a barrier to remain attached to the desired site.
Many small animal models (mice, rats, rabbits) commonly described in the literature are acceptable analogues of the biochemical processes involved in adhesion formation. Valuable information on adhesion formation, reformation and prevention has been obtained from these models. However, when compared with humans, these models have differences in scale, surgical procedure, organ size, organ weight and physiological forces. These differences can reduce the ability of these models to predict clinical success.
To this end, some have proposed large animal models such as a canine radical pelvic resection model that simulates radical hysterectomy (Montz et al., 1993b), a porcine incisional hernia repair model (Christoforoni et al., 1996
), a porcine pelvic surgery model (Montz et al., 1993a
) and a ewe hysterotomy model that simulates a myomectomy (Moll et al., 1992
). No large animal models have been adequately described for the formation of adnexal adhesions with the pelvic sidewall.
In this study we present a porcine model for adhesion formation following tubal and ovarian surgery. An attempt is made to develop site-specific adhesions with high reproducibility using a surgical procedure analogous to adnexal surgery or myomectomy.
This model creates typical surgical conditions encountered in tubal and ovarian surgery, a clinically relevant procedure that is known to be at high risk for the development of post-operative adhesions. The inclusion of several adhesiogenic stimuli that are routine during surgery, such as de-peritonealization of tissues and ischaemic insults from electrocautery and sutures, creates a surgical environment optimal for evaluating adhesion barriers. In this manner, preclinical animal model testing of proposed prophylactic anti-adhesion materials can more accurately predict ultimate clinical efficacy in humans. In addition to developing this animal model, the present study also evaluated a specific adhesion barrier material, SprayGel.
Relative to the control side, there was a 67% reduction in the incidence of adhesions at the treated side. On the treated side, only the pelvic sidewall received the adhesion barrier, leaving the injured uterine horn and bladder unprotected. It is significant to note that adhesions were observed on the treated side between the uterine horn and the bladder, demonstrating that the barrier did indeed exclude the adhesiogenic horn from the sidewall, but not from untreated ipsilateral sites.
High inter-animal variability in animal models has led to the suggestion of using each animal as its own control (Ordonez et al., 1997). This of course can only be performed with non-regional adhesion prevention strategies. Since in this study the adhesion barrier is applied locally by spraying and does not redistribute throughout the pelvis like a liquid or viscous gel, such a model can be used resulting in distinct statistical advantages. Moreover, the need for internal controls is particularly important when using large animals not specifically bred for genetic similarity.
Despite the presence of several large animal adhesion formation models in the literature, there is a need for a reliable large animal model of adhesion formation following tubal and ovarian surgery. Given the histological similarities between porcine uterine horns and human Fallopian tubes, this model seems appropriate to assess post-operative adhesions following adnexal surgery.
This porcine model also allowed the creation of conditions that are relevant to the human surgical environment in terms of organ size and forces exerted on the adhesion barrier. These conditions are needed to better evaluate important barrier features such as ease of placement and resistance to migration from the desired site.
In order to obtain consistent adhesions at the control site, it was determined during model development that the injury process needed to be performed via laparotomy. Therefore, the adhesion barrier was applied during the same open procedure. In the future, laparoscopic material application following the laparotomy closure may be performed. Even though in this model the adhesion barrier was not deployed laparoscopically, the sprayable nature of this barrier allows for the easy deposition in both laparoscopic and open surgical scenarios. The presence of a colourant allows for an easy visualization of the hydrogel and precise placement. The transformation of the precursor solutions to a tissue adherent hydrogel takes place within seconds and large denuded or traumatized areas can be expeditiously protected. An undetectable amount of heat is liberated during the gelation process, and due to the high water content of the components a lubricious surface is presented to surrounding tissues and organs after deposition.
PEG is a poor substrate for bacteria due to its non-biological origin. Thus, along with the rapid barrier absorption rate (less than 1 week), the barrier material does not lend itself readily to the promotion or potentiation of bacterial infection. Despite the emergence of several regional adhesion prevention instillates, there is a clear need for an efficacious, easy to use site-specific adhesion barrier that can be used laparoscopically. Thus, SprayGel will potentially address the need of the laparoscopic surgeon who needs to protect site-specific injuries that are susceptible to post-surgical adhesion formation.
The results of this study lead us to conclude that this porcine model of adhesion formation is appropriate for the investigation of site-specific adhesion formation and prevention in a clinically relevant surgical procedure. The promising efficacy demonstrated by the PEG adhesion barrier in this and other (Dunn et al., 2001) models of adhesion formation warrant the further investigation of this adhesion barrier material in a larger animal study.
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Acknowledgements |
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Notes |
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Statement of commercial interest
D.Mulani is a former employee while P.K.Campbell is a current employee of Confluent Surgical Inc. R.Ferland is a consultant to Confluent Surgical Inc.
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References |
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Submitted on October 27, 2000; accepted on August 28, 2001.