Abstract
Objective: To compare the host and biomechanical response to a fully absorbable poly-4-hydroxybutyrate (P4HB) scaffold to the response to PP mesh in an animal model of vaginal POP surgery.
Design: A study employing a sheep model
Setting: KU Leuven Center for Surgical Technologies
Population: 14 parous female Mule sheep
Methods : P4HB scaffolds were surgically implanted in the posterior vaginal wall of sheep. The comparative PP mesh data were obtained from an identical protocol.
Main outcome measures: Gross necropsy, histological and biomechanical evaluation of explants, and the in vivo P4HB scaffold degradation were evaluated at 60- and 180-days post-implantation.
Results: Gross necropsy revealed no implant related adverse events using P4HB scaffolds. The tensile stiffness of the P4HB explants increased at 180-days (12.498 ± 2.66 N/mm (P=0.019)) as compared to 60-days (4.585 ± 1.57 N/mm) post-implantation, while P4HB degraded gradually. P4HB scaffolds exhibited excellent tissue integration with dense connective tissue and a moderate initial host response. P4HB scaffolds induced a significantly higher M2/M1 ratio (1.70 ± 0.67 SD, score 0-4), as compared to PP mesh (0.99 ± 0.78 SEM, score 0-4) at 180-days.
Conclusions: P4HB scaffold facilitated a gradual load transfer to vaginal tissue over time. The fully absorbable P4HB scaffold, in comparison to PP mesh, has a favorable host response with comparable load bearing capacity. If these results are also observed at longer follow-up, a clinical study for vaginal POP surgery may be warranted to demonstrate efficacy.
Key words: Pelvic organ prolapse, vaginal surgery, Poly-4-hydroxybutyrate, degradable scaffold, host response, biomechanics.
INTRODUCTION
Pelvic organ prolapse (POP) is a common condition resulting from damage to the supportive structures of the pelvic floor(1, 2). The annual incidence of surgery for POP is approximately 4.9 cases per 1000 women with the overall life-time risk for POP surgery of 11%(3, 4). Synthetic permanent polypropylene (PP) meshes have been introduced to surgical repair of POP to provide mechanical support to the pelvic floor by inducing a foreign body response(3, 5). However, they have been associated with the clinical complications in long-term. Even though, PP meshes have been modified(6, 7) and resulted milder host response and better outcomes(8), the reputation is damaged. The US Food and Drug Administration (FDA) re-classified transvaginal POP meshes from Class II to Class III in 2016 and have not approved vaginal PP implants since April 2019 in some countries including the USA. The Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) (2015) has recommended identification of alternatives to polypropylene, and focusing on biodegradable biomaterials for POP repair to reduce the risk of long-term complications(9).
Our research group has identified poly-4-hydroxybutyrate (P4HB) as a candidate material for vaginal POP surgery(10) with the hypothesis that a delayed-absorbable implant will provide mechanical support while being gradually replaced by functional connective tissue. P4HB is a biologically produced biosynthetic polymer(11) which degrades to the human metabolite 4HB(12) and gets eliminated from the body completely. Several P4HB devices for soft tissue support have been cleared by the FDA(13). P4HB has also been used for many other clinical applications, including reconstructive surgery, tendon, and ligament repair.
Knitted P4HB scaffold, provide good anatomical and functional outcomes in hernia repair(14), although the applied forces are different, it still concerns a load-bearing soft tissue correction, as in the case of POP. Our previous in vitro study illustrated that vaginal fibroblasts on P4HB scaffolds generated a more favourable cellular proliferation, and collagen deposition than on PP(10). In addition, the knit design favouring the optimal cellular response to P4HB scaffold was identified.
These previous outcomes encouraged us that P4HB was a promising candidate material for pelvic floor surgery. Therefore we, decided to further evaluate the host response and biomechanics of fully degradable P4HB scaffold in sheep, an animal model for vaginal POP surgery(15, 16) The outcomes of P4HB scaffold were compared to data of PP mesh obtained from an identical study performed by our group.
  1. MATERIAL and METHODS
  2. ImplantsBased on our previously performed in vitro studies (10), we selected a knitted, monofilament P4HB scaffold design with an implant thickness of 0.28 mm, a fibre diameter of 100 µm, and a pore size of 2.22 mm2. As comparison, light-weight polypropylene – Restorelle® (Coloplast, Minneapolis, MN, USA), with an implant thickness of 0.34 mm, a fibre diameter of 80 µm, and a pore size of 3.1 mm2, was used.
  3. Animals, surgical procedures and study design
Animals, anaesthesia and surgical procedures are detailed in Supplementary material 1 and 2. The animals used in this study were maintained and treated according to experimental protocols (P057/2014//P 064/2013and P051/2016) approved by the Ethics Committee on Animal Experimentation of the Faculty of Medicine, KU Leuven. 14 parous female Mule sheep (7 years old, weighing 51.5 ± 5.7 kg) were randomly divided into two groups for each time point. All sheep underwent rectovaginal surgery for the implantation of P4HB scaffold. The surgical procedure was carried out according to the previously described method (4) by the same experienced surgeon (LH). Briefly, the rectovaginal septum was dissected following hydro-dissection. P4HB scaffolds (35 x 35 mm) were fixed with interrupted non-degradable 3/0 polypropylene sutures (Prolene®, Ethicon, Zaventem, Belgium) at the corners and halfway along each side (Figure 1). The vaginal wall was closed with a running 3/0 polyglactin 910 (Vicryl) suture.
Harvesting ImplantsEwes were premedicated by intramuscular administration of 1 ml/50 kg xylazine and euthanised with intravenous pentobarbital (20 ml/50 kg Release, Ecuphar, Oostkamp, Belgium)(17). Gross anatomical examination of the explanted vagina was performed using the following parameters: i) fluid collection, ii) exposure of the implant, iii) synechiae, iv) signs of infection. Any shrinkage of the implant was calculated by measuring the length and width of the scaffold. Vaginal explants (vaginal tissue/P4HB implant complex) were dissected into four pieces for assessing both active and passive biomechanical properties, in vivo degradation of the P4HB scaffold, and histomorphology.
Figure 1.
  1. Outcome measurementsBefore implantation
  2. Mechanical properties of the implant before the implantation Before the implantation, P4HB scaffold and PP mesh were subjected to a uniaxial testing under dry conditions according to a standardised protocol (18). Details of the uniaxial testing was explained in supplementary material 3.After implantation
  3. Active biomechanical properties Longitudinal vaginal strips (3 x 7 mm) from explants were dissected, weighed, and immediately suspended in individual organ baths containing fresh Krebs solution (4). Samples were pre-tensioned to 0.5 mN and equilibrated for 60 min before measurement. The samples were subjected to contractile stimulation by 80 mM of KCl. Contractile forces were recorded using custom-made software. Measurements were analysed using Origin software (OriginLab Corporation, Northampton, MA, USA). All values were normalised to sample weight, transducer calibration and gravitation constant.
  4. Passive biomechanical properties
Uni-axial tensiometry on the vaginal explants was performed by using Zwick uni-axial tensiometer (Zwick GmbH & Co. KG, Ulm, Germany) with a 200N load cell. Posterior middle vaginal tissue was used as a control. Samples were cut longitudinally (10 x 30 mm), clamped tension free, and the zero elongation was defined as the clamp-to-clamp distance at preload (0.1 N). The samples were loaded to failure with an elongation rate of 10 mm/min. The strain was calculated by dividing the elongation by the clamp-to-clamp distance and stress dividing force by the cross-sectional area (19). The stiffness (N/cm2) of the specimens was determined with the slope of the stress-strain curve in comfort zone by using TestXpert II software (Zwick GmbH & Co) (18).
In vivo degradation of the P4HB implant
In vivo degradation of P4HB scaffolds were determined by molecular weight (Mw) change via gel permeation chromatography (GPC) analysis and by changes in scaffold morphology via scanning electron microscopy (SEM) (JEOL JSM6700F) following to vaginal tissue by digestion.(20). Details of the tissue digestion, and analysis of GPC and SEM were explained in supplementary material 4.
Histomorphology Details of histology and immunohistochemistry (IHC) staining, and scoring is given at Supplementary material 5 (Table S1, Figure S1). Tissue integration of the P4HB scaffolds was evaluated from SEM images of the vaginal explants. Histology sections were stained with Haematoxylin and Eosin (H&E) and Masson Trichrome to quantify the foreign body giant cells (FBGC), polymorphonuclear cells (PMN), blood vessels and connective tissue. IHC staining was performed for detection of neovascularisation (CD34), neuronal network (PGP 9.5), myofibroblasts and smooth muscle cells (α-SMA), leukocytes (CD45), M1 (HLA-DR) and M2 (CD163) macrophages. The M2/M1 ratio was calculated. Semi-quantitative assessment was performed by using a qualitative grading scale (4, 21) by two individual researchers blinded for both timepoints.
Statistical analysis
Statistical analysis was performed with GraphPad Prism 7.0 (GraphPad Software, Inc; La Jolla, USA). Data normality was tested by the Kolmogorov-Smirnov test. Two-way ANOVA was used for normally distributed data and multiple comparisons between individual groups using a Tukey’s test. The Kruskal-Wallis followed by the Dunn’s post hoc test was used for data that was not normally distributed. Data are reported as mean ± standard deviation or median and standard error of the mean as appropriate. The significance level was defined as p <0.05.
RESULTS
3.1. Before Implantation