Expression of Vascular Endothelial Growth Factor and Interleukin-6 in bile duct healing with autologous parietal peritoneum: a non-inferiority experimental study in rabbits

Animal model

An animal study was conducted with approval from the Institutional Review Board of the Ethics Committee of the Dr. Moewardi General Hospital, Surakarta, Indonesia with the number of 1.785/X/HREC/2023. The study included adult male New Zealand rabbits (Oryctolagus caniculus), aged 8–10 months and weighing between 2.0–3.0 kg. The New Zealand rabbits used in this study were obtained from the Prof. Soeparwi Veterinary Hospital, Faculty of Veterinary Medicine, Gadjah Mada University, an accredited breeding facility that adheres to ethical guidelines for animal care and use. Rabbits that were not in good condition were excluded based on predefined criteria. The minimum sample size was determined using Federer’s Formula, with at least nine rabbits per group. 41 rabbits that initially met our inclusion criteria underwent surgical procedures. However, 11 rabbits (26.82%), consisting of three from the control group, three from the parietal peritoneum group, and five from the vesica fellea group, were excluded from the study due to death before anastomosis tissue collection. The causes of death varied, but autopsy findings indicated that bile duct leakage was the primary cause in most cases.

Intervention groups

Rabbits were randomly divided into three groups of nine rabbits each. Group A (n = 9) act as a control and had the common bile duct primarily closed. Group B (n = 9) underwent cholecystectomy (1.5 cm length), insertion of a five cm tube as a stent, and transplantation of the parietal peritoneum. Group C (n = 9) had a cholecystectomy (1.5 cm length), insertion of a five cm tube as a stent, and transplantation of the gallbladder.

Intervention procedure

1. Pre-operative procedure. Following the Johns Hopkins University’s Animal Care and Use Committee (JHU ACUC) standards, rabbits were allowed to adapt to their new environment for 2–3 days before the surgical procedure. Rabbits were housed in individual cages with a minimum area of 0.5 square meters per animal, in a temperature-controlled environment (27 °C), with a 12-hour light/dark cycle. They were fed a standard rabbit diet consisting of pelleted food and provided with fresh water ad libitum. Blood samples were taken prior to surgery to assess blood chemistry. For pre-operative preparation, the rabbits were given albendazole at a dose of 20–50 mg/kg body weight (BW) orally to prevent worms and were fasted for 4–8 h before surgery. To further minimize confounding variables, all rabbits were kept in a controlled environment at a constant temperature of 27 °C. Additionally, their diets were standardized, and cage sizes were uniform to ensure that external factors did not influence the outcomes of the study.

2. Anesthetic procedure. According to Johns Hopkins University Animal Care and Use Committee (JHU ACUC) standards, rabbits were anesthetized with intravenous ketamine (50 mg/kg) and dexilasane (1–2 mg/kg), along with a 0.9% NaCl infusion. Ventilation was maintained with 2% halothane inhalation and 100% oxygen. The rabbits were positioned in a supine decubitus position, with anesthesia maintained using isoflurane and sevoflurane through a vaporizer.

3. Biliary-digestive-repair. After anesthetization, the rabbits were shaved, subjected to antisepsis, and placed in a sterile field. A 5-cm midline abdominal incision was made from the xiphoid process to the peritoneal cavity to expose and identify the common bile duct (CBD). A 15 mm × 5 mm excision was performed on the anterior wall of the CBD to create a defect. A similar excision was made on the parietal peritoneum harvested from the posterior abdominal wall to repair the defect (Figs. 1A, 1B). The graft was positioned over the CBD, and a 5-cm plastic stent (3.5 Fr NGT) was carefully inserted into the lumen to maintain patency (Fig. 1C). The biliodigestive anastomosis was secured using a 7-0 polypropylene simple interrupted suture, ensuring proper graft integration. The final anastomotic site showed a well-positioned graft with no immediate complications upon completion of the procedure (Fig. 1D).

4. Postoperative care. Postoperatively, the rabbits were given standard food and had free access to water, along with a 0.9% NaCl infusion. Intravenous atipamezole (five mg/kg) and xylazine (five mg/kg) in a 1:1 ratio were administered. The rabbits were kept in clean cages at a room temperature of 27 °C.

5. Animal termination. Rabbits were humanely euthanized on days 3, 7, and 14 post-treatment using an intravenous injection of phenobarbital at a dose of 100 mg/kg, following institutional ethical guidelines and the approved protocol. No rabbits required early euthanasia or met any criteria for premature termination. In cases where a rabbit died prematurely, it was excluded from the study. Biliary anastomosis tissue was taken to be examined by enzyme-linked immunosorbent assay (ELISA) for levels of IL-6 and VEGF. To ensure unbiased results, the laboratory analysts performing the ELISA, as well as all laboratory personnel involved, were blinded to the group assignments and specimen control. This blinding was strictly maintained throughout the analysis to prevent any potential bias in the evaluation of wound healing markers across the different treatment groups.

Immunoassay

The ELISA examination used the sandwich enzyme-linked immunosorbent assay (ELISA) method to quantify IL-6 and VEGF levels in tissue samples. The Rabbit IL-6 ELISA kit (MBS2507990, MyBioSource, Indonesia) and Rabbit VEGF ELISA kit (MBS765626, MyBioSource, Indonesia) followed the manufacturer’s protocol.

Sample preparation: tissue samples were cut, weighed, and homogenized in phosphate-buffered saline (PBS) (pH 7.4) at 4 °C. The homogenate was centrifuged at 14,000 rpm for 20 min, and the supernatant was collected for analysis.

Assay plates were prepared by adding 100 µL of standards or samples to designated wells, including a 0 pg/mL standard diluent as a negative control. The wells were sealed and incubated at 37 °C for 90 min. After incubation, biotinylated detection antibodies (100 µL per well) were introduced and incubated at 37 °C for 60 min. Following three washing cycles, 90 µL of horseradish peroxidase (HRP)-conjugate solution was added per well and incubated for 30 min at 37 °C. The plate was washed five times, and 100 µL of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution was added to initiate a colorimetric reaction. After 20 min of incubation at 37 °C in the dark, 100 µL of stop solution was added, changing the color from blue to yellow.

Optical density (OD) values were measured at 450 nm using an ELISA reader, and IL-6 and VEGF concentrations were determined based on standard curves.

This study employed a true experimental post-test-only control group design to investigate the effect of autologous parietal peritoneum grafts on biliary tract injury repair. Experiments were conducted between February and April 2023.

Fibroblast

The assessment of fibroblast activity was carried out through histological examination, utilizing hematoxylin and eosin (H&E) staining to observe tissue remodeling and healing processes in bile duct repair sites. To preserve both cellular and extracellular structures, tissue specimens were initially fixed in 10% buffered formalin for 24–48 h. Following fixation, the samples underwent a graded ethanol dehydration process (70%, 80%, 95%, and 100%), followed by xylene clearing to eliminate residual lipids and impurities. The specimens were then embedded in paraffin wax, creating stable blocks for further sectioning.

For microscopic evaluation, thin sections (five µm) were obtained from the paraffin-embedded samples using a microtome and then mounted onto clean glass slides. The slides were subjected to deparaffinization in xylene, after which they were progressively rehydrated through a series of descending ethanol concentrations (100%, 95%, 80%, and 70%). A final rinse in distilled water restored their hydrophilic properties. Hematoxylin staining was employed to highlight cell nuclei in blue, while eosin staining provided contrast by coloring the cytoplasm and extracellular matrix in shades of pink.

Microscopic observations were conducted under 400 × magnification using a light microscope. To quantify fibroblast density, fibroblasts were counted in five randomly selected high-power fields (HPFs) per sample, and the average number per HPF was determined as an indicator of tissue remodeling and wound healing. To ensure reliability, all slides were examined independently by two pathologists, and any discrepancies in fibroblast identification were resolved through collaborative evaluation and consensus discussions. Additionally, a qualitative histopathological analysis was performed to further assess tissue responses at the repair site.

Histopathology

The surviving rabbits underwent an exploratory laparotomy, during which the entire extrahepatic biliary system, including both grafts and native bile ducts, was excised. The collected bile duct specimens were promptly fixed in 10% neutral buffered formalin to preserve tissue integrity. The specimens were sent to the Pathology Department at Gadjah Mada University for histopathological evaluation. Hematoxylin and eosin (H&E) staining was conducted to assess the bile ducts’ morphological structures, providing detailed insight into tissue architecture and potential pathological changes.

Statistical analysis

All statistical analyses were conducted using SPSS version 28.0 (IBM Corp., New York, USA). The Shapiro–Wilk test was used to assess data normality, while Levene’s test evaluated variance homogeneity.

For normally distributed data with equal variances, one-way analysis of variance (ANOVA) was performed, followed by Bonferroni post-hoc analysis for pairwise comparisons. If data did not follow a normal distribution, the Kruskal–Wallis test was applied, with Dunn’s post-hoc test for multiple comparisons. When normality was met but variance homogeneity was violated, the Brown-Forsythe ANOVA was used, followed by Dunnett T3 post-hoc analysis to adjust for unequal variance.

Although sample size determination was based on Federer’s Formula, ensuring a minimum of nine rabbits per group, the study was primarily powered to detect early differences in IL-6, VEGF expression, and fibroblast activity. The statistical analysis included tests for normality and variance homogeneity, with non-parametric methods applied when necessary to maintain statistical robustness and ensure valid comparisons.

Parametric data were expressed as mean ± standard deviation (SD), while non-parametric data were presented as median and interquartile range (IQR). Statistical significance was determined using two-tailed tests, with a p-value <0.05 considered significant. Where applicable, effect sizes were calculated to provide a clearer understanding of the differences between groups.

VEGF

A significant increase in VEGF signals angiogenesis, enhancing wound tissue perfusion and increasing oxygen availability (PO₂), which supports cell proliferation and tissue repair (Shaik et al., 2020; Setiawan et al., 2022). In bile duct repair, VEGF plays a specialized role in promoting biliary epithelial cell (BEC) proliferation and modulating VEGFR-2 signaling, which facilitates interactions between inflammatory and mesenchymal cells, contributing to ductal healing (Mariotti et al., 2021).

Persistent inflammation can impair angiogenesis by increasing reactive oxygen species (ROS), activating NF-κB, suppressing VEGF expression, and inducing apoptosis (caspase-3) (Pereira Beserra et al., 2019; Mussbacher et al., 2023). In our study, ductal VEGF levels on days 3, 7, and 14 did not differ significantly between groups (p > 0.05), suggesting that all repair strategies provided comparable angiogenic support (Kim & Byzova, 2014). However, VEGF levels were higher in the autologous peritoneum group, likely due to inosculation-driven vascularization, where angiogenesis facilitates graft integration.

While VEGF is essential for biliary epithelial regeneration, excessive neovascularization can promote fibrosis and bile duct stricture formation, as observed in chronic biliary diseases (Mariotti et al., 2021). This highlights the delicate balance between sufficient angiogenesis for healing and excessive vascular remodeling, which may increase the risk of stenosis. Future studies should evaluate the long-term effects of VEGF-driven vascular remodeling in biliary anastomotic healing and fibrosis prevention (Mussbacher et al., 2023; Kim & Byzova, 2014; Johnson et al., 2020).

IL-6

IL-6 is a key regulator of inflammation and tissue remodeling, orchestrating immune cell recruitment, angiogenesis, and extracellular matrix modulation (Johnson et al., 2020). In bile duct repair, IL-6 plays a dual role: at controlled levels, it promotes biliary epithelial cell (BEC) proliferation and ductal remodeling, but prolonged IL-6 activity can lead to fibrosis and bile duct stricture formation (Bester & Pretorius, 2016).

IL-6 exerts its effects through JAK/STAT3 and NF-κB pathways, which regulate biliary epithelial survival, collagen deposition, and inflammatory resolution (Li et al., 2022). The presence of IL-6 and its receptor in human BECs suggests that IL-6 may exert both autocrine and paracrine effects, directly influencing biliary epithelium regeneration and fibrotic progression (Demetris et al., 2006).

In our study, ductal IL-6 levels on days 3, 7, and 14 did not show significant differences between groups (p > 0.05), suggesting that all repair strategies maintained a controlled inflammatory response. However, IL-6 levels appeared higher in the autologous peritoneum group, possibly reflecting a localized early inflammatory response that may have facilitated immune cell infiltration and wound remodeling. While this response could be beneficial for early epithelial repair, excessive IL-6-driven inflammation has been implicated in biliary fibrosis and chronic inflammation (Li et al., 2022).

This highlights the need for precise regulation of IL-6 activity—sufficient to drive bile duct healing but controlled to prevent fibrosis. Future studies should focus on IL-6 modulation strategies to optimize early inflammatory responses while mitigating long-term fibrotic progression, ensuring effective bile duct repair without pathological scarring.

IL-6 is a crucial regulator in wound healing, managing inflammatory responses, promoting tissue regeneration, aiding angiogenesis, and ensuring proper tissue remodeling. Its ability to balance pro-inflammatory and anti-inflammatory signals is vital for efficient and effective wound repair (Johnson et al., 2020). IL-6 signaling affects vascular integrity through endothelial activation, increased vascular permeability, immune cell recruitment, and vascular wall fibrosis. These effects reduce nitric oxide (NO) availability, as IL-6 influences endothelial nitric oxide synthase activity and expression while increasing superoxide production, which deactivates NO, affecting coagulation and platelet profiles (Bester & Pretorius, 2016; Didion, 2017). In the study, ductal IL-6 levels on days 3, 7, and 14 did not show significant differences between the treatment groups (p > 0.05), suggesting that excessive inflammation was absent in any groups. Although IL-6 levels appeared higher in the autologous parietal peritoneum group compared to the control, this trend did not reach statistical significance. This may indicate a potential early inflammatory response, which could facilitate immune cell recruitment and tissue repair, but further investigation is needed to confirm this hypothesis. However, while IL-6 plays a beneficial role in early healing, prolonged and excessive IL-6 expression could lead to chronic inflammation and fibrosis, highlighting the need for balanced inflammatory responses in effective healing (Johnson et al., 2020; Soliman & Barreda, 2023).

Fibroblast

The significantly lower number of fibroblasts in the autologous parietal peritoneum group on day 3 (p = 0.019) likely reflects an initial delay in fibroblast recruitment. This delay may be attributed to reduced fibrinolytic activity following peritoneal trauma, which impairs the conversion of plasminogen to plasmin, delaying fibrin degradation and slowing early wound healing processes. Additionally, delayed mesothelial cell regeneration—which plays a key role in initiating fibrinolytic activity from the wound bed—further contributes to this delay (Bakkum et al., 1996). Neutrophils produce Tumor Necrosis Factor (TNF)-α and Interleukin (IL)-1, which play a role in recruiting epithelial cells and fibroblasts to the wound area. The increase in fibroblast numbers occurs due to stimulation from fibroblastic growth factors (FGF), which are secreted as proteins during the platelet degranulation process (Goldberg & Diegelmann, 2010). By day 7, the number of fibroblasts increased in all groups, consistent with the proliferative phase of wound healing, indicating active tissue repair. Although the differences between groups were not significant, the steady increase in the autologous parietal peritoneum group suggests progressive integration and healing. During the proliferation phase, connective tissue and granulation tissue are formed due to fibroblast activation. Along with this, reepithelialization, neovascularization, and immunomodulation occur (Rodrigues et al., 2019). On day 14, the decrease in fibroblast numbers in the control group may indicate the onset of the remodeling phase, while the continuously higher numbers in the autologous parietal peritoneum and vesica fellea groups suggest ongoing tissue repair.

Histological analysis

The histological findings provide key insights into the healing dynamics of autologous peritoneum grafts in bile duct repair. The significantly lower fibroblast infiltration observed on day 3 suggests delayed initial recruitment, likely due to reduced fibrinolytic activity and slower mesothelial regeneration following peritoneal trauma. However, by day 7, fibroblast density increased, indicating a transition to the proliferative phase, characterized by granulation tissue formation and extracellular matrix remodeling.

By day 14, fibroblast distribution stabilized across all groups, with no significant intergroup differences, suggesting that the initial delay in fibroblast infiltration in the autologous peritoneum group did not impair long-term tissue remodeling. The presence of well-organized tissue architecture at this stage indicates that the graft successfully integrated and facilitated progressive healing. These findings suggest that autologous peritoneum grafts provide a controlled regenerative environment, promoting gradual tissue remodeling while potentially minimizing excessive fibroblast proliferation that could contribute to fibrosis and ductal stenosis. Thus, despite a slightly delayed early response, autologous peritoneum appears to support gradual and effective bile duct healing, reinforcing its potential as a viable graft material for biliary reconstruction.