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.