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\begin{document}

\title{EVALUATION OF THE SHORT TERM HOST RESPONSE AND BIOMECHANICS OF AN
ABSORBABLE POLY-4-HYDROXYBUTYRATE SCAFFOLD IN A SHEEP MODEL FOLLOWING
VAGINAL IMPLANTATION}



\author[1]{Chantal Diedrich}%
\author[1]{Zeliha Guler}%
\author[2]{Lucie Hajkova Hympanova}%
\author[1]{Eva Vodegel}%
\author[3]{Manuel  Zundel}%
\author[3]{Edoardo Mazza}%
\author[4]{Jan Deprest}%
\author[1]{Jan-Paul Roovers}%
\affil[1]{Amsterdam UMC Locatie AMC}%
\affil[2]{KU Leuven}%
\affil[3]{ETH Zurich}%
\affil[4]{Center for Surgical Technologies}%


\vspace{-1em}



  
  \date{June 13, 2024}


\begingroup
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\selectlanguage{english}
\begin{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 \selectlanguage{ngerman}± 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.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{EVALUATION OF THE SHORT TERM HOST RESPONSE AND BIOMECHANICS OF
AN ABSORBABLE POLY-4-HYDROXYBUTYRATE SCAFFOLD IN A SHEEP MODEL FOLLOWING
VAGINAL IMPLANTATION}

\textbf{P4HB scaffold induces vaginal tissue remodelling}

\selectlanguage{greek}\emph{Chantal M. Diedrich\textsuperscript{1γ}, Zeliha
Guler\textsuperscript{1γ}} \selectlanguage{english}\textsuperscript{*}\emph{,} Lucie
Hympanova\textsuperscript{2,3}, Eva Vodegel\textsuperscript{1}, Manuel
Z\selectlanguage{ngerman}ündel\textsuperscript{4}, Edoardo Mazza\textsuperscript{4}, Jan
Deprest\textsuperscript{2}, Jan Paul Roovers\textsuperscript{1}

\textsuperscript{1} Department of Obstetrics and Gynaecology, Amsterdam
Reproduction and Development, Amsterdam UMC -- location AMC, University
of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands

\textsuperscript{2} Centre for Surgical Technologies, Group Biomedical
Sciences, KU Leuven, Leuven, Belgium

\textsuperscript{3} Institute for the Care of Mother and Child, Third
Faculty of Medicine, Charles University, Prague, Czech Republic

\textsuperscript{4} Institute of Mechanical Systems, ETH Zurich, Zurich,
Switzerland; i EMPA, Swiss Federal Laboratories for Materials Science
and Technology, Dübendorf, Switzerland

\selectlanguage{greek}γ \selectlanguage{english}These authors contributed equally and should be considered as first
co-authors

*Corresponding author: Z. Guler, Department of Obstetrics and
Gynaecology, Amsterdam Reproduction and Development, Amsterdam UMC --
location AMC, University of Amsterdam, Meibergdreef 9, Amsterdam, The
Netherlands, email: zeliha.guler@amsterdamumc.nl, Tel: +31 20 5666199

\textbf{Abstract}

\textbf{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.

\textbf{Design:} A study employing a sheep model

\textbf{Setting:} KU Leuven Center for Surgical Technologies

\textbf{Population:} 14 parous female Mule sheep

\textbf{Methods} : P4HB scaffolds were surgically implanted in the
posterior vaginal wall of sheep. The comparative PP mesh data were
obtained from an identical protocol.

\textbf{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.

\textbf{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 \selectlanguage{ngerman}± 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.

\textbf{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.

\textbf{Key words:} Pelvic organ prolapse, vaginal surgery,
Poly-4-hydroxybutyrate, degradable scaffold, host response,
biomechanics.

\textbf{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 \emph{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.

\begin{enumerate}
\tightlist
\item
  \textbf{MATERIAL and METHODS}
\item
  \textbf{Implants}Based on our previously performed \emph{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 \selectlanguage{greek}µ\selectlanguage{english}m, and
  a pore size of 2.22 mm\textsuperscript{2}. As comparison, light-weight
  polypropylene -- Restorelle\selectlanguage{ngerman}® (Coloplast, Minneapolis, MN, USA), with
  an implant thickness of 0.34 mm, a fibre diameter of 80 \selectlanguage{greek}µ\selectlanguage{english}m, and a pore
  size of 3.1 mm\textsuperscript{2}, was used.
\item
  \textbf{Animals, surgical procedures and study design}
\end{enumerate}

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 \selectlanguage{ngerman}± 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.

\textbf{Harvesting Implants}Ewes 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.

\begin{enumerate}
\tightlist
\item
  \textbf{Outcome measurementsBefore implantation}
\item
  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.\textbf{After implantation}
\item
  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.
\item
  Passive biomechanical properties
\end{enumerate}

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 (\selectlanguage{greek}α-\selectlanguage{english}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.

\textbf{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 \selectlanguage{ngerman}±
standard deviation or median and standard error of the mean as
appropriate. The significance level was defined as p \textless{}0.05.

\textbf{RESULTS}

\textbf{3.1. Before Implantation}

3.1.1. Mechanical characteristics of the implants

P4HB scaffold and PP mesh exhibited similar behaviour under cyclic
uniaxial load (Figure 2) with a strong inelastic deformation in the
first cycle, followed by a fast stabilisation. However, the membrane
stiffness of the PP mesh is threefold higher compared to P4HB scaffold
within ten cycles. A decrease in the stiffness of the PP mesh was
observed after 10 cycles contrary to the P4HB scaffold which exhibited a
relatively small increase in its stiffness after cyclic mechanical
loading (17).

\textbf{Figure 2.}

\textbf{3.2. After Implantation}

3.2.1. Gross anatomical examination of the explanted vagina and the
implant contraction

All implants were well incorporated in the deeper vaginal tissue layers
without any signs of encapsulation. We did not observe any exposure of
P4HB scaffolds or fluid collection at 60- and 180-days
post-implantation. Sheep implanted with PP mesh (n=6) developed vaginal
synechiae (n=3). P4HB scaffolds were intact, without macroscopic
deformation. Shrinkage (\% area) of the implants after 60 days and 180
days were 52.2 ± 10.41 SD and 45.9 ± 7.95 SD, respectively. The
shrinkage of the PP implants (29.6±7.8 SD and 46.7±14.2 SD) at 60 and
180 days post-implantation were reported as this previously (4).

3.2.2. Active biomechanical properties

Vaginal tissue contraction (Figure 3) in response to KCl was higher at
180 days (226.90 mN ± 79.08, SEM) in comparison to 60 days
post-implantation (148.53 mN ± 22.76, SEM). Vaginal tissue exhibited
higher contractile function after P4HB scaffold implantation compared to
after PP mesh implantation (37.87 mN ± 18.99, SEM at 60-day and 199.49
mN ± 71.56, SEM at 180-day), however the difference at both time points
was not statistically significant.

\textbf{Figure 3} .

3.3.3. Passive biomechanical properties

Biomechanical properties of the vaginal explants and control tissue were
compared at 60- days and 180- days post-implantation (Figure 4). The
P4HB explants at 60 days, exhibited significantly lower stiffness values
compared to the control tissue (4.58 ± 1.57 N/mm SEM vs 11.49 SEM,
p=0.024). The stiffness of the explants after 180 days post-implantation
increased significantly (12.498 ± 2.66 N/mm, 0.019) as compared to
60-days post-implantation and exhibited a comparable stiffness with the
control tissue (11.343 ± 1.96 N/mm, SEM). According to the ball burst
test which is not directly comparable to uni-axial tensile testing,
there were no statistically significant changes in stiffness values of
the PP explants at both time-points (4).

\textbf{Figure 4.}

3.3.4. In vivo degradation of the P4HB scaffold

The degradation of P4HB scaffold was determined by change in molecular
weightover time (Figure 5A). The average Mw of the P4HB scaffold
gradually decreased significantly over time, 279 ± 3 kDa (SD), 201 ± 5
kDa (SD) and 104 ± 7 kDa (SD) at 0-, 60- and 180- days
post-implantation, respectively. The integrity and morphological changes
of the implant was visualised by SEM (Figure 5B). The monofilament of
P4HB scaffold with smooth surface pre-implantation kept its overall
macroscopic integrity at both time-points. SEM images revealed the
formation of fibre surface erosion over time. At 60 days, there were
only superficial scratches on the fibres, which gradually progressed
into surface fissuring over 180 days post-implantation.

\textbf{Figure 5.}

3.3.5. Histomorphology

P4HB Scaffold maintained its integrity after 60- and 180-days post
implantation and showed good integration within the submucosa of the
vaginal tissue (Figure 6). The P4HB scaffold could be identified in
between the lamina propria and muscularis layer in the H\&E-stained
samples (Figure 7). There was no significant difference in the presence
of FBGCs at the border of the implant between both time points (0.93 ±
0.42 SD at 60-days and 0.73 ± 0.54 SD at 180-days) (Figure 8A). A
decrease in the presence of polymorphonuclear cells (PMNCs) was observed
over time, however this difference was not significant (2.06 ± 0.70 SD
at 60-days and 1.65 ± 0.83 SD at 180-days) (Figure 8B). There was
significantly higher inflammatory cell infiltration around the P4HB
scaffold as compared to the PP mesh at both 60- (P4HB: 2.06 ± 0.70 and
PP: 0.60 ±0.31 SD) and 180-days (P4HB: 1.65 ± 0.83 and PP:0.32 ±0.10 SD)
(Figure S2A) (4). There was no significant difference in vessel count
around the P4HB scaffold at 60- and 180-days based on H\&E staining
(Figure 8C). However, PP mesh, exhibited a higher number of vessels as
compared to P4HB scaffold at both time points (P4HB: 0.87 ± 054 SD at
60-days, 0.78 ± 0.53 SD at 180-days; PP: 1.52 ± 0.46 SD at 60-days and
1.96 ± 0.32 SD at 180-days) (Figure S2B). At 60 days, P4HB scaffolds
were surrounded with newly formed connective tissue with aligned
collagen fibres. After 180 days the connective tissue matured, indicated
with darker blue, and density increased (Figure 8D).

\textbf{Figure 6.}

\textbf{Figure 7.}

\textbf{Figure 8.}

There was no significant difference of neovascularisation (Figure 9B)
and neuronal ingrowth (Figure 9F) and myofibroblast differentiation
(Figure 9H) between 60- and 180-days after vaginal implantation with
P4HB scaffold. Presence of leukocytes at 180 days (score 2.7 ± 0.86 SD)
was significantly higher compared to 60 days (score 2.05 ± 0.55 SD)
after P4HB scaffold implantation (Figure 9D). At 180-days a more
widespread infiltration area was CD45 positive, whereas at 60-post
implantation this was more visible at the implant border. When compared
to PP mesh, P4HB scaffold exhibited significantly (P\textless{}0.005)
higher leukocyte infiltration (PP=1.29 ± 0.36 SD) at 60 days (Figure
S3A), and significantly lower myofibroblast differentiation (PP: 2.41 ±
0.26 and P4HB: 1.62 ± 0.56 SD) at 180 days (Figure S3B).

\textbf{Figure 9.}

There was a statistically significant decrease observed in macrophage
type-1 (M1) infiltration to P4HB scaffold between implantation times of
60- and 180-days (Figure 10B). At 60 days, M1 infiltration was more
dominant (2.02 ± 0.62 SD) at the P4HB scaffold-tissue interface compared
to 180 days (1.56 ± 0.59 SD). No statistically significant difference in
M2 infiltration was observed between 60- (1.97±0.85 SD) and 180-days
(2.24 ± 0.66 SD) post-implantation of P4HB scaffold. The M2/M1 ratio was
significantly (P\textless{}0.01) higher at 180-days (1.70 ± 0.67 SD) as
compared to 60-days (0.91± 0.60 SD) post-implantation of P4HB scaffold
(Figure 10F). If compared with the retrospective PP mesh data, M2/M1
ratio at 180-days post-implantation was significantly higher with the
use of P4HB scaffold (Figure S3C). (PP 0.92 ± 0.17 SD and 0.99 ± 0.78 SD
at 60- and 180-days, respectively).

\textbf{Figure 10.}

\textbf{DISCUSSION}

This study is the first preclinical evaluation of P4HB scaffold in
vaginal prolapse surgery using a large animal (sheep) model. We have
evaluated the host inflammatory response, biomechanical properties, and
degradation profile of explants over time. Here we did not observe
scaffold exposure, or any other adverse events such as infection, fluid
collection or synechiae at 60- and 180-days post-implantation. The
stiffness of the vaginal P4HB scaffold explants increased between 60-
and 180-days, while the polymer underwent significant degradation over
time. P4HB scaffold resulted in a moderate host response, which is
demonstrated by an increased M2/M1 ratio (remodelling), low
myofibroblast differentiation and formation of well-organised collagen
over time, compared to the PP mesh.

In our current study, we obtained well-remodelled vaginal tissue with
enhanced biomechanical properties after transvaginal implantation of
P4HB scaffold into sheep, which serve as a good translation model for
women (4, 15, 22). The host response in terms of vascularisation and
collagen deposition after P4HB scaffold implantation were similar to the
effect of lightweight PP mesh. However, the initial inflammatory
response of P4HB scaffold was greater compared to PP mesh. After
180-days the inflammatory state changed to a more calm and chronic state
of the host tissue and resulted in tissue remodelling (macrophage type 2
response). Inflammatory reaction to slowly degrading hybrid implant
(TIGR® Matrix) also got milder over time and resulted a thicker tissue
formation, as compared to PP mesh, in a sheep abdominal hernia model
(23). The P4HB scaffold were still intact and well-integrated with the
vaginal tissue, without signs of encapsulation or exposure, something
that was seen with the use of PP mesh. This might be related to the
exceptionally low membrane stiffness of the P4HB scaffold. Constant
applied load after implantation can cause an increase of the implant
stiffness, which, in turn, may lead to vaginal degeneration and
eventually exposure (24, 25). One can predict no or limited exposures
due to the P4HB scaffold, as even after 10 cycles of mechanical loading,
there was only a slight increase in the stiffness of the P4HB implants.
Gradual absorption of P4HB provided a good balance between implant
degradation and new tissue formation. Fast degradation of the implants
may be associated with poor clinical outcomes due to the weakness of the
remodelled tissue. For instance, it was reported that degradable
polyglactin-910 implants disappeared within a 6-week period after
anterior vaginal wall implantation (26), which resulted in 25\% of
recurrent cystoceles after 12 months. De Tayrac at al. (27) found that
the PLA implant lost most of its mass already after 1.5 months \emph{in
vitro,} where degradation is slower compared to in vivo. Apart from
preclinical studies for synthetic degradable materials, there are
examples of clinically used degradable xenografts in transvaginal
surgery such as porcine dermis or porcine small intestinal submucosa
such as InteXEN® (28) and Surgisis® (29). However, due to rapid
degradation of xenografts, the load bearing capacity of vaginal tissue
after the surgery was insufficient in the long term due which resulted
in recurrent prolapse (28). Therefore, a sufficient degradation profile
of a newly designed implant is necessary to maintain pelvic floor
support over time. In the case of P4HB Scaffold, contrary to the
decrease in molecular weight, the stiffness of the vaginal explants was
increased over time, explants had the comparable stiffness with the
vaginal tissue. The stiffness of the explants were at least 10 times
higher even when compared to the initial stiffness of the P4HB Scaffold,
which suggests remodelling and regeneration of the vaginal tissue
contributes to increased tissue stiffness (23). The tissue components
such as collagen contribute to the biomechanical properties of the
tissue by allowing the tissue to resist deformation under mechanical
force (30). As the absorbable P4HB scaffold slowly and gradually
degrades, it induced functional, viable vaginal tissue with mechanical
integrity. The functionality of the vagina, which can be determined by
its ability to actively contract by the smooth muscle activity, may
change after implantation. The presence of an implant may induce
fibrosis, or alter the collagen or elastin content which subsequently
result in a decrease in the vaginal contractility (22). Despite the
increase in stiffness of the P4HB scaffold explants, and no difference
on the mechanical properties of the PP mesh vaginal explants over time,
the contractile function of the vagina implanted with P4HB scaffold was
higher than the ones with PP mesh. Implantation of P4HB scaffold
resulted in a higher number of inflammatory cells compared to PP mesh,
which might be attributed to a higher areal density of P4HB scaffold
compared to PP mesh (4) or the ongoing degradation of the P4HB scaffold.
However, cellular infiltration of inflammatory cells within the P4HB
scaffold/tissue interface was decreased over time and was followed by
organised connective at the later stage. The increase in CD45 positive
cells at 180 days might be caused by an inflammatory process towards a
more chronic state of the foreign body response with a transient
presence of monocytes, FBGCs and perhaps lymphocytes (31). Implant
properties influence the inflammatory cell interactions at the
implant-tissue interface and lead to altered foreign body responses, may
further promote greater biocompatibility upon implantation(32). Hjort
et.al reported a moderate and decreased inflammatory response over time
due to gradual implant degradation(23). If we look at the M2/M1 ratio,
P4HB Scaffolds were dominated by M2 macrophages compared to the PP mesh
at 180-days post-implantation. Transition from M1 to M2 phenotype occurs
with the initiation of the remodelling phase of wound healing and
enhanced tissue regeneration could be expected (33, 34). In addition,
myofibroblast differentiation, which plays a key role in the existence
of fibrosis (35, 36), was less pronounced in the P4HB scaffold explants
at 180-days compared with PP mesh. This finding also supports the result
of a mechanically stronger tissue which is created by the effective
remodelling process after P4HB scaffold implantation.

We acknowledge some limitations of this study. We have used
retrospective data for PP mesh, as the design of the current study was
identical to the previous. This approach is in line with the aim of
reducing the use of animals in research (37). The duration of the study
is not sufficient to demonstrate the tissue properties after complete
absorption of P4HB scaffold. To characterize the long-term performance
of P4HB Scaffold, a longer-term follow-up is necessary.

On the other hand, this is the first preclinical study demonstrating the
performance of P4HB Scaffold as a transvaginal implant. We used a well-
established animal model used in pelvic floor research. Additionally,
the P4HB scaffold was well-characterised in our recent study (10) before
transvaginal implantation.

P4HB scaffold exhibits good mechanical support to vaginal tissue and
results in a moderate host response in vivo without any visible implant
related complications. Although six-month data shows gradual load
transfer from the P4HB scaffold to the vaginal tissue, the biomechanics
of the tissue need to be further evaluated after complete degradation to
determine if restored vaginal tissue strength and stiffness is
self-sufficient to withstand the loads of daily life. Based on these
encouraging results, we have started a two-year follow up study in
sheep, prior to introducing the P4HB scaffold clinically. The P4HB
scaffold may represent a unique fully absorbable alternative to
permanent polypropylene mesh for the surgical correction of POP in
women.

\textbf{Disclosure of interests}

CMD., ZG., LH, EV, MZ, EM and JD declare that they have no competing
interests. JPR declares that he received unrestricted research grants of
Coloplast, Tepha Inc.

\textbf{Contribution to authorship}

JPR and JD planned and developed the study. CMD and LH performed the
implantations, and CMD, LH, EV and ZG performed the explantations and
sample collection. ZG, CMD, EV, LH, MZ, EM performed data collection and
analysis. ZG was the primary author of the manuscript, and all authors
revised the manuscript and approved the final version.

\textbf{Details of ethics approval}

This study was conducted with the approval (LA1210191, approval date
January 2016) of the the Ethics Committee on Animal Experimentation of
the Faculty of Medicine, KU Leuven and in adherence to experimental
protocols (P057/2014//P 064/2013and P051/2016).

\textbf{Funding}

Financial contribution was provided by Tepha, Inc., Lexington, MA, USA
with unrestricted research grant.

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