Feasibility of New Zealand rabbit as an animal model for the study of biological grafts in pelvic reconstructive surgery

Peró Garcia M1, Porta Roda O2, Alserawan De Lamo L1, Canovas Noé M3, Casani Arazo L3, Castells Sala C4, Florit Carbonell S3, Grandal Rejo B1, Juan Babot J3, Longo A1, Lopez Chicon P4, Minovas Granja G1, Moga Naranjo M1, Pérez Rodríguez M4, Tur Tur V1

Research Type

Basic Science / Translational

Abstract Category

Pelvic Organ Prolapse

Abstract 355
E-Poster 2
Scientific Open Discussion ePoster Session 18
Thursday 5th September 2019
13:05 - 13:10 (ePoster Station 7)
Exhibition Hall
Animal Study Basic Science Grafts: Biological Pre-Clinical testing
1.Hospital de la Santa Creu i de Sant Pau, 2.Hosp, 3.Institut de Recerca de l'Hospital de la Santa Creu i de Sant Pau, 4.Banc de Sang i Teixits Barcelona

Marta Peró Garcia




Hypothesis / aims of study
Mesh reinforced repair is currently a standard procedure in pelvic reconstructive surgery. Prosthetic materials are usually synthetic and unabsorbable. As synthetic mesh materials are associated with a risk of severe complications, there is a growing interest in finding better materials that help improve outcomes.

Our research group studied the properties of a biological graft material, a human acellular dermal matrix (hADM) to be used in reconstructive pelvic surgery by testing it in an animal model. hADM is an extracellular matrix developed by Barcelona Tissue Bank in which the starting material is human cadaveric skin that undergoes a decellularization process in order to remove the cellular components of the tissue leaving the extracellular matrix intact. This matrix was compared to an unabsorbable, synthetic equivalent mesh graft to study how both grafts behaved after implantation in abdominal and vaginal surgery. The results will help to define hADM characteristics with respect to its capacity to successfully integrate in vivo, thereby guiding the design of novel materials for this challenging clinical application.

Animal studies are necessary to examine biomechanical properties of the implanted grafts and to determine the tissue response in order to evaluate safety and efficacy of different proposed materials. Most studies to develop new biomaterials use mice as animal model. However, we wished to study implants in a vaginal location to mimic as close as possible the conditions found in pelvic reconstructive surgery. Therefore, we sought an animal model that allowed vaginal surgery and could be easily barned. Little information exists regarding the characteristics of different animal models when used for vaginal surgery.

The objective was to evaluate the New Zealand (NZ) rabbit as an animal model for testing a hADM as a biomaterial to be used in pelvic reconstructive surgery.
Study design, materials and methods
Twenty NZ rabbits were randomized into 2 groups: 10 received hADM grafts while the remaining 10 were implanted with polypropilene (PP) mesh. Each rabbit had 4 segments implanted: 2 into the abdomen and 2 in the vagina, in the submucosal layer. The graft segments were removed 180 days later. Surgical findings and clinical results were analyzed.

Rabbits were anesthetized with ketamine (15 mg/Kg subcutaneous) and Medetomidine (0.5 mg/Kg subcutaneous). Each animal received antibiotic prophylaxis with a cefalosporine (Ceftiofur 50 mg/Kg subcutaneous) and a pain killer (Meloxicam 1mg/Kg subcutaneous). Animals were carefully shaved prior to surgical incision. Also prior to surgery and at day 7, 30 and 180, 6cc of blood were extracted from the auricular artery for the study of inflammatory markers purposes. Total operative time was recorded.

Rabbits were strictly monitored until euthanasia was performed at 180 days. Signs of loss of wellbeing were looked after. Pain was assessed with the Grimace scale. Any sign of macroscopic surgical site infection was guarded. After sacrifice of animals, grafts were explanted to perform different analyses: macroscopic findings (i.e. erosion), biomechanical, immunohistochemical and histological. Biomechanical properties of abdominal and vaginal explants (both elasticity and stiffness) was planned to be assessed by uniaxial tensiometry, using as control groups the biomaterials before its implantation.

Comparisons between categorical variables were calculated by chi-square test with the approximation of likelihood ratio. When appropriate, the comparison between two groups was made with the Mann-Whitney nonparametric test. In all cases the level of significance was set at the usual 5% (alpha = 0.05). All analysis were performed with the IBM-SPSS (V25) statistical package.
Surgical findings and clinical data follow-up are described in Table 1.

The average operative time was 75 minutes in the control group, and 80 minutes in the experimental group (p = 0.807).

The most relevant surgical difficulties during the implantation procedure were: adequate exposure of the vaginal surgical field, because of the small size; the maintenance of the integrity of the vaginal mucosa layer, because of its extreme thinness; the maintenance of adequate aseptic conditions, because of the high amount of animal hair; and the location and dissection of the lateral thoracic vein during abdominal surgery.

Blood extraction was difficult in 6/20 cases (30%). There were no significant differences between groups (p = 0.08).

The most serious complication was death of one animal in the control group, 58 days after surgery. The necropsy showed no complications at the mesh level, nor other pathological findings of interest.

Table 1 shows clinical complications, that were significantly more common in the control group (70%) vs the experimental group (20%) (p=0.021, likelihood ratio).

All individuals experimented a progressive weight gain. The mean weight increase in the control group was 884 g, while in the experimental group it was 714.5 g (p=0.641).

The explantation surgery was performed in 19 of the 20 individuals (95%). Table 1 shows the most relevant pathological findings. A greater number of abnormal findings during explantation of grafts was observed in the control group (60%) compared with the experimental group (10%) (p= 0.015). However, macroscopic degradation of the vaginal grafts was more common in the experimental group (40%), while in the control group the vaginal mesh was identified in 100% of the individuals (p = 0.01). 

The mean vaginal pH at the time of sacrifice in the control group was 8.47, and in the experimental group it was 8.38 (p=0.173).
Interpretation of results
The placement of grafts in the abdomen and in the vagina of NZ rabbits is feasible, technically simple, and it's not associated to serious surgical complications. However, the adequate exposure of the vaginal field is difficult, due to animal's small size. This limitation was overcome with the help of an assistant and adequate material.

The blood extraction from the auricular artery can be difficult, especially after successive extractions in the same individual. Therefore, it is advisable to have the help of personnel trained in this technique.

Clinical complications associated with the grafts (wound infection and extrusion) are more common in the control (mesh) group. However, in the experimental group the macroscopic degradation of the matrix occurs at the vaginal level and may impair its correct identification to proceed with biomechanical testing. Instead, it remains without macroscopic changes in its abdominal location.

In both groups there are self-injuries due to stereotypes, so it is recommended to add environmental enrichment measures in these animals. It is also important to maintain strict hygiene measures to avoid complications derived from dirt.
Concluding message
It is feasible to use New Zealand rabbits as an animal model to reproduce surgeries both abdominally and vaginally to test grafts. Although this model comes with some difficulties related to the small size of the animal, it is compensated by the benefits of the model: it is an easily acquired animal, which offers the researcher a quick learning curve regarding its management and caring, and presents a cost-efficient barning.
Figure 1 Table 1. Surgical findings and clinical follow-up data
Funding This project has received funding thanks to a competitive announcement from the Banc de Sang i Teixits de Barcelona (BST). Agreement BST - IIB Sant Pau 2017-3: "Study of the biological properties of a human dermal matrix for its application in surgery to correct prolapse of pelvic organs and female urinary incontinence", with € 150,594.40 (divided into 3 annuities). From 1/7/2017 to 6/30/2020 Clinical Trial No Subjects Animal Species New Zealand race rabbit Ethics Committee The study was approved by the Animal Experimentation Ethics Committee and by government authorities (FUE-2017-00561151, Project number 9669).