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 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.