The development of anastomotic leak (AL), defined as a defect of the intestinal wall at the anastomotic site leading to a communication between the intra- and extra-luminal compartments,¹ is one of the most feared complications following colorectal resection surgery. Patients who experience AL are at an increased risk of negative patient outcomes including infection or sepsis, cancer recurrence, and post-operative mortality.^2–8 Although variable rates of AL have been reported in the literature, it has been shown to occur in 2.4%–10%^2,3,7,9–14 of cases. A number of patient-related and peri-operative factors have been implicated as risk factors for AL, including male sex,^2,5,9,10,13 tobacco use,^10,13,14 pre-operative serum albumin,^2,12,13 longer operative duration,^10,12 open surgical procedures,^5,14 obesity,^2,10 and diabetes.^5,13

Patients who experience AL not only exhibit worse post-operative outcomes and have a higher risk of mortality, but they also incur healthcare costs that are approximately three-fold higher than patients without a leak.^7,8,15 In one study involving nearly 100,000 patients, ALs resulted in $24,129 of additional hospital costs within the first hospitalization.⁷ The economic burden associated with ALs—including hospitalization and re-admission—was $28.6 million in additional costs per 1,000 patients undergoing colorectal surgery.⁷ Elevated healthcare costs are partially due to increased length of stay in the hospital,^2,3,5–8 which is more than doubled for patients with an AL, as well as an increased rate of complications and hospital re-admission.^3,7,14 As a result of the high morbidity and economic burden, there remains an unmet need for enhancing anastomotic closure to improve healing and prevent post-operative leakage.

In recent years, amniotic membrane (AM) derived from the placenta and umbilical cord (UC) have been increasingly used as a biologic dressing to promote healing and restore function due to its anti-inflammatory and anti-scarring properties.^16–18 While few clinical studies have assessed the benefit of ultra-thick AM from the UC for ALs, a number of pre-clinical animal model studies have demonstrated the benefit of AM from the placenta in preventing AL in rats, dogs, and rabbits.^19–24 In several rat model studies, rats who received AM over colonic anastomoses demonstrated significantly higher bursting pressure, collagen deposition, and hydroxyproline concentrations at days 3 and 7 compared to the control group, suggesting higher intrinsic resistance of anastomoses to rupture.^19,20,25 Furthermore, inflammatory cell infiltration and adhesion scores were significantly lower in the AM group compared to controls,^19,20,25 and rates of anastomotic dehiscence/leak were lower at day 7 in rats receiving AM compared to controls (5.0% vs. 42.1%, respectively).²⁰ In another rat animal model study, AL was observed in only one rat with AM wrapping and in all 28 rats without AM wrapping.²⁴ Similarly, lower rates of colorectal anastomosis dehiscence have been demonstrated in rabbits treated with AM, with 0% (0/13) of AM-treated rabbits showing no leakage after 8 weeks compared to 71% (5/7) of rabbits in the control group.²³ Given this promising pre-clinical data, we sought to assess the safety and effectiveness of ultra-thick AM graft in preventing AL in patients undergoing intestinal surgery.

A retrospective chart review was conducted on patients who underwent colorectal and intestinal surgery with adjunctive cryopreserved, ultra-thick AM derived from the UC (Clarix^® 1K; BioTissue, Miami FL), between January 2016 and January 2023 by a single surgeon. The study was conducted at a single center in accordance with the tenets of the Declaration of Helsinki and was exempt under 45 CFR §46.104(d)(4) by the Sterling Institutional Review Board. Data collected from the medical charts included demographic information, significant medical history including co-morbidities and prior surgeries, indication and location of resection, length of removed colon, surgical duration, time to first defecation, length of hospital stay (days), additional treatments and medications, and complications.

The patient was taken to the operating room and after successful endotracheal intubation, the patient was placed in lithotomy position. A skin incision was made after administration of local anesthetic and blunt dissection was carried down to the abdominal wall fascia. The fascia was opened up and dissection was used to enter the peritoneum. The intestines area of interest were mobilized and transected with an endostapler. The 2 ends were brought together in an end-to-end or side-by-side anastomosis was created. The colon was approximated with multiple interrupted 3-0 silk sutures. The open portion of the colon was then brought together and then stapled off with a TA 30 stapler x2 (Medtronic). An inspection was then made to ensure there was no leak seen along either portion of the staple line. Ultra-thick AM (Clarix^® 1K; BioTissue) was then placed over the anastomosis and secured down with multiple 2-0 Vicryl sutures. A second ultra-thick AM was placed on the posterior aspect of the anastomosis and secured down with multiple 2-0 Vicryl sutures (Fig. 1). The bowel was placed back in its anatomical position and the abdomen was irrigated with saline as well as Irrisept. The fascia was then closed with a #1 looped polydioxanone suture. The wound was irrigated with saline, the fascia was closed in a figure-of-eight fashion, and the skin was approximated with a interrupted 3-0 Vicryl suture. Dressings were applied and the patient was awoken, extubated, and taken to the recovery room.

Figure 1. Representative example of surgical placement of the ultra-thick amniotic membrane. A 74-year-old Caucasian male that had multiple risk factors including former smoker, heart failure, and chronic obstructive pulmonary disease underwent laparoscopic-assisted low anterior resection surgery for diverticulitis. During the procedure, 140 mm colon was resected followed by end-to-end anastomosis. Two 6 cm × 3 cm ultra-thick amniotic membranes (A) were sutured together lengthwise (B) and then sutured over the anastomosis site (C).

Eleven consecutive patients (7 male, 4 female) fulfilled the eligibility criteria and were included in this study. At the time of surgery, patients had an average age of 66.5 ± 10.9 years (47–84 years) and an average body mass index of 27.7 ± 4.2 kg/m². All patient demographics are provided in Table 1. Patients underwent open (n = 2) or laparoscopic (n = 9) intestinal reconstructive procedure for diverticulitis (n = 4), colon mass (n = 2), cecal polyp/mass (n = 2), bowel erosion (n = 1), rectal prolapse (n = 1), and colostomy reversal (n = 1) involving the sigmoid (n = 6), cecum (n = 2), ascending colon (n = 1), rectum (n = 1), and small bowel (n = 1). The average length of intestine removed was 120.5 ± 62.1 mm, which was followed by end-to-end or side-by-side single layer anastomosis and placement of ultra-thick AMs (two 6 cm × 3 cm units in 6 cases; two 8 cm × 3 cm units in 3 cases; or one 8 cm × 3 cm unit in 2 cases). Surgery was uneventful in all cases and was completed in an average duration of 98.5 ± 40.0 min.

Table 1 . Demographics of Study Cohort.

Characteristic	Value (n = 11)
Age (yr)	66.5 ± 10.9
Body mass index (kg/m2)	27.7 ± 4.2
Sex
Female	4 (36.4)
Male	7 (63.6)
Race, white	11 (100)
Ethnicity, Hispanic	2 (18.2)
Smoker
Yes	2 (18.2)
No	6 (54.5)
Former	3 (27.3)
Comorbidities
Obesity	2 (18.2)
Hypertension	2 (18.2)
Diabetes	1 (9.1)
Heart failure	1 (9.1)
Chronic obstructive pulmonary disease	1 (9.1)
Prior abdominal surgery	2 (18.2)

Values are presented as mean ± standard deviation or number (%)..

Post-operatively, the average length of hospital stay was 6.7 ± 7.3 days (median: 4 days, range: 3–28 days), and the time to first defecation was 3.5 ± 2.1 days (median: 3 days). Nine patients took narcotics (hydrocodone/acetaminophen) post-operatively to help manage post-surgical pain. After an average follow up of 65.3 ± 42.0 days (median: 70 days), none of the patients had suffered an AL nor required another colorectal procedure despite the use of chemotherapy in one patient. One 75-year-old patient succumbed to atrial fibrillation 28 days after the procedure. Other complications included mild ileus (n = 1) and difficulty urinating (n = 1).

Despite the development of less invasive surgical techniques, AL still remains a high-risk complication following colorectal and intestinal surgery. While the exact etiology of AL remain unclear, there is accumulating evidence suggesting AL is multifactorial and the result of several factors and biological processes, including inflammation.²⁶ In this retrospective review, we assessed the clinical effectiveness and safety of cryopreserved, ultra-thick AM as an anastomotic wrap in facilitating healing and functional recovery in patients who underwent colorectal and intestinal surgery. Importantly, no cases of AL were observed post-operatively in this cohort of patients who were predominantly male and had history of smoking. These findings are promising given male sex,^2,5,9,10,13 and history of tobacco use^10,13,14 are significant risk factors of post-operative AL. Moreover, the median time to first defecation was 2.5 days, and the median hospital length of stay was only 4 days. Comparatively, one report involving 8,197 patients found the median length of stay was 14 days for elective surgery and 21 days for emergency admissions following colorectal resection.²⁷ Furthermore, the median time to first bowel movement has been reported to occur 3–5 days post-operatively with conventional care.^28–30 Collectively, this preliminary data suggests that use of cryopreserved, ultra-thick AM as an anastomotic wrap may potentially expediate healing, time to first defecation and the incidence of AL.

While a number of different grafting materials have been used to reinforce the anastomotic region and prevent leakage, many have proven to be unsuccessful in pre-clinical models. In a rat model, both human dura mater and free peritoneal graft reinforcement groups had worse anastomosis healing compared to the control group, as supported by significantly lower bowel bursting pressure, more adhesions, and presence of anastomotic stricture.³¹ In another study, rats who received collagen fleece had significantly more bowel obstructions and more severe inflammation compared to the control group, with no significant differences observed between groups in anastomotic bursting pressures and complications.³² Similarly, rabbits with polyglycolic acid mesh reinforced anastomosis demonstrated an increased risk of AL and peritoneal adhesion as well as decreased collagen fiber density and neovascularization compared to controls.³³ Furthermore, anastomoses sealed by non-cross-linked collagenous matrix in a rodent model showed impaired anastomotic healing, as demonstrated by increased abdominal adhesions and decreased blood vessel ingrowth, collagen deposition, neovascularization, and bursting pressure.³⁴ Not only have the aforementioned biomaterials proven unsuccessful in improving anastomotic healing, they have also demonstrated significantly worse outcomes, suggesting the need for a graft that is both immune-privileged and reduces inflammation and scarring to promote regenerative healing. AM is one such grafting material that has been shown to promote healing of colorectal anastomoses in a number of pre-clinical animal model studies as demonstrated by significantly lower rates of AL, inflammatory cell infiltration, and adhesion as well as significantly greater bursting pressures, collagen deposition, and hydroxyproline concentrations compared to controls.^19–24 Although the clinical data is scarce, one retrospective clinical study has assessed the use of dehydrated AM and chorion in reducing the rate AL in humans. In that study, Ortega et al³⁵ found that 4 of 390 (1.03%) anastomoses wrapped with dehydrated human amnion/chorion membrane leaked compared to 80 of 2,000 (4.0%) in the control group.

The potential utility of ultra-thick AM in supporting healing and reducing AL may be attributed to the anti-inflammatory and anti-scarring properties inherent to AM and UC tissues.^36–38 One key complex present in cryopreserved, ultra-thick AM tissues, heavy chain-hyaluronan/pentraxin 3 (HC-HA/PTX3), has been shown to exhibit immunomodulatory properties through its ability to promote apoptosis of activated neutrophils and macrophages^36,37,39,40 and promote polarization of M1 macrophages to the M2, anti-inflammatory phenotype,^37,39 which is critical in resolving inflammation and facilitating wound healing.^41–43 Moreover, such an anti-inflammatory effect extends to adaptive immune responses by HC-HA/PTX3’s ability to suppress CD4+ T cell activation and downregulate alloreactive immune responses.^39,44 While suppression of inflammation indirectly reduces scarring, there is strong evidence to demonstrate that HC-HA/PTX3 directly reduces scarring by inhibiting phosphorylated Smad nuclear translocation, suppressing transforming growth factor-beta1 promoter activity, preventing α-smooth muscle actin expression, and reducing myofibroblast differentiation.^36,45–50 Together these conditions may provide an optimal environment to support scarless healing, which may reduce the occurrence of strictures and adhesions and ultimately the risk of AL.

While this preliminary data is encouraging, this study is limited by its small sample size and variable follow-up times due to the study’s retrospective design. While several patients had shorter follow-up times, previous studies have shown that AL, on average, occurs within 8.6–12 days^3,51,52 post-operatively. Furthermore, patients are more inclined to return to the clinic when complications arise, suggesting these patients who failed to return for subsequent follow-up visits recovered without complication. Prospective, randomized, controlled trials are necessary to further ascertain the benefit of ultra-thick AM and to determine whether its use significantly reduces the incidence of AL and complications post-operatively.

These preliminary findings suggest cryopreserved, ultra-thick AM is safe and may help prevent AL and post-operative complications following colorectal and intestinal surgery. Larger prospective, randomized, controlled trials are warranted.

None.

Data is available from the author upon reasonable request.

No potential conflict of interest relevant to this article was reported.