Uncovering the relationship between macrophages and polypropylene surgical mesh.

Currently, in vitro testing examines the cytotoxicity of biomaterials but fails to consider how materials respond to mechanical forces and the immune response to them; both are crucial for successful long-term implantation. A notable example of this failure is polypropylene mid-urethral mesh used in the treatment of stress urinary incontinence (SUI). The mesh was largely successful in abdominal hernia repair but produced significant complications when repurposed to treat SUI. Developing more physiologically relevant in vitro test models would allow more physiologically relevant data to be collected about how biomaterials will interact with the body. This study investigates the effects of mechanochemical distress (a combination of oxidation and mechanical distention) on polypropylene mesh surfaces and the effect this has on macrophage gene expression. Surface topology of the mesh was characterised using SEM and AFM; ATR-FTIR, EDX and Raman spectroscopy was applied to detect surface oxidation and structural molecular alterations. Uniaxial mechanical testing was performed to reveal any bulk mechanical changes. RT-qPCR of selected pro-fibrotic and pro-inflammatory genes was carried out on macrophages cultured on control and mechanochemically distressed PP mesh. Following exposure to mechanochemical distress the mesh surface was observed to crack and craze and helical defects were detected in the polymer backbone. Surface oxidation of the mesh was seen after macrophage attachment for 7 days. These changes in mesh surface triggered modified gene expression in macrophages. Pro-fibrotic and pro-inflammatory genes were upregulated after macrophages were cultured on mechanochemically distressed mesh, whereas the same genes were down-regulated in macrophages exposed to control mesh. This study highlights the relationship between macrophages and polypropylene surgical mesh, thus offering more insight into the fate of an implanted material than existing in vitro testing.

Developing Wound Dressings Using 2-deoxy-D-Ribose to Induce Angiogenesis as a Backdoor Route for Stimulating the Production of Vascular Endothelial Growth Factor.

2-deoxy-D-Ribose (2dDR) was first identified in 1930 in the structure of DNA and discovered as a degradation product of it later when the enzyme thymidine phosphorylase breaks down thymidine into thymine. In 2017, our research group explored the development of wound dressings based on the delivery of this sugar to induce angiogenesis in chronic wounds. In this review, we will survey the small volume of conflicting literature on this and related sugars, some of which are reported to be anti-angiogenic. We review the evidence of 2dDR having the ability to stimulate a range of pro-angiogenic activities in vitro and in a chick pro-angiogenic bioassay and to stimulate new blood vessel formation and wound healing in normal and diabetic rat models. The biological actions of 2dDR were found to be 80 to 100% as effective as VEGF in addition to upregulating the production of VEGF. We then demonstrated the uptake and delivery of the sugar from a range of experimental and commercial dressings. In conclusion, its pro-angiogenic properties combined with its improved stability on storage compared to VEGF, its low cost, and ease of incorporation into a range of established wound dressings make 2dDR an attractive alternative to VEGF for wound dressing development.

A novel characterisation approach to reveal the mechano-chemical effects of oxidation and dynamic distension on polypropylene surgical mesh.

Polypropylene (PP) surgical mesh, used successfully for the surgical repair of abdominal hernias, is associated with serious clinical complications when used in the pelvic floor for repair of stress urinary incontinence or support of pelvic organ prolapse. While manufacturers claim that the material is inert and non-degradable, there is a growing body of evidence that asserts PP fibres are subject to oxidative damage and indeed explanted material from patients suffering with clinical complications has shown some evidence of fibre cracking and oxidation. It has been proposed that a pathological cellular response to the surgical mesh contributes to the medical complications; however, the mechanisms that trigger the specific host response against the material are not well understood. Specifically, this study was constructed to investigate the mechano-chemical effects of oxidation and dynamic distension on polypropylene surgical mesh. To do this we used a novel advanced spectroscopical characterisation technique, secondary electron hyperspectral imaging (SEHI), which is based on the collection of secondary electron emission spectra in a scanning electron microscope (SEM) to reveal mechanical-chemical reactions within PP meshes.

Author Correction: Regenerative medicine and injection therapies in stress urinary incontinence.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regenerative medicine and injection therapies in stress urinary incontinence.

Stress urinary incontinence (SUI) is a common and bothersome condition. Anti-incontinence surgery has high cure rates, but concerns about mesh tapes have resulted in the resurgence of surgical procedures that involve increased abdominopelvic dissection and morbidity. Injection therapy with urethral bulking agents or stem cell formulations have been developed as minimally invasive alternatives. Many synthetic and biological bulking agents have been trialled, but several have been discontinued owing to safety concerns. The use of Macroplastique and Contigen has the largest evidence base, but, overall, success rates seem to be similar between the various agents and positive outcomes are poorly sustained for more than 6 months. Furthermore, subjective cure rates, although initially high, also deteriorate over time. The available data consistently demonstrate manifestly poorer outcomes for injection therapies than for surgery. Stem cell treatments are thought to functionally regenerate the urethral sphincter in patients with suspected intrinsic sphincter deficiency. Autologous adipose and muscle-derived stem cells seem to be the intuitive cell source, as they are comparatively abundant, can be harvested and cause minimal donor site morbidity. To date, only a few small clinical studies have been reported and most data are derived from animal models. The success rates of stem cell injection therapies seem to be comparable with those of bulking agents.

Complications related to use of mesh implants in surgical treatment of stress urinary incontinence and pelvic organ prolapse: infection or inflammation?

The surgical mesh material used in the surgical treatment of stress urinary incontinence (SUI) and pelvic organ prolapse (POP) in women is associated with significant complications in some women. This has recently become a public health issue with involvement of national parliaments and regulatory bodies. The occurrence of mesh complications is thought to be a result of multifactorial processes involving problems related to the material design, the surgical techniques used and disease, and patient-related factors. However, the infectious complications and mesh-tissue interactions are least studied. The aim of this article is to review any previous clinical and basic scientific evidence about the contribution of infectious and inflammatory processes to the occurrence of mesh-related complications in SUI and POP. A literature search for the relevant publications without any time limits was performed on the Medline database. There is evidence to show that vaginal meshes are associated with an unfavourable host response at the site of implantation. The underlying mechanisms leading to this type of host response is not completely clear. Mesh contamination with vaginal flora during surgical implantation can be a factor modifying the host response if there is a subclinical infection that can trigger a sustained inflammation. More basic science research is required to identify the biological mechanisms causing a sustained inflammation at the mesh-tissue interface that can then lead to contraction, mesh erosion, and pain.

Assessment of Electrospun and Ultra-lightweight Polypropylene Meshes in the Sheep Model for Vaginal Surgery.

BACKGROUND: There is an urgent need to develop better materials to provide anatomical support to the pelvic floor without compromising its function. OBJECTIVE: Our aim was to assess outcomes after simulated vaginal prolapse repair in a sheep model using three different materials: (1) ultra-lightweight polypropylene (PP) non-degradable textile (Restorelle) mesh, (2) electrospun biodegradable ureidopyrimidinone-polycarbonate (UPy-PC), and (3) electrospun non-degradable polyurethane (PU) mesh in comparison with simulated native tissue repair (NTR). These implants may reduce implant-related complications and avoid vaginal function loss. DESIGN, SETTING, AND PARTICIPANTS: A controlled trial was performed involving 48 ewes that underwent NTR or mesh repair with PP, UPy-PC, or PU meshes (n=12/group). Explants were examined 60 and 180 d (six per group) post-implantation. INTERVENTION: Posterior rectovaginal dissection, NTR, or mesh repair. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Implant-related complications, vaginal contractility, compliance, and host response were assessed. Power calculation and analysis of variance testing were used to enable comparison between the four groups. RESULTS: There were no visible implant-related complications. None of the implants compromised vaginal wall contractility, and passive biomechanical properties were similar to those after NTR. Shrinkage over the surgery area was around 35% for NTR and all mesh-augmented repairs. All materials were integrated well with similar connective tissue composition, vascularization, and innervation. The inflammatory response was mild with electrospun implants, inducing both more macrophages yet with relatively more type 2 macrophages present at an early stage than the PP mesh. CONCLUSIONS: Three very different materials were all well tolerated in the sheep vagina. Biomechanical findings were similar for all mesh-augmented repair and NTR. Constructs induced slightly different mid-term inflammatory profiles. PATIENT SUMMARY: Product innovation is needed to reduce implant-related complications. We tested two novel implants, electrospun and an ultra-lightweight polypropylene textile mesh, in a physiologically relevant model for vaginal surgery. All gave encouraging outcomes.

Developing a synthetic composite membrane for cleft palate repair.

An oronasal fistula is a passage between the oral and nasal cavity. Currently, surgical procedures use mucosal flaps or collagen grafts to make a barrier between oral and nasal cavities. Our aim was to develop a cell-free synthetic repair material for closure of nasal fistulas. We surface functionalized electrospun polyurethane (PU) and poly-L-lactic acid (PLLA) and composite polymer (PU-PLLA) membranes with acrylic acid through plasma polymerization. Membranes were treated in a layer-by-layer approach to develop highly charged electrostatic layer that could bind heparin as a pro-angiogenic glycosaminoglycan. The properties were evaluated through physical, chemical, and mechanical characterization techniques. Cytotoxicity was tested with MC3T3 pre-osteoblast cell lines for 3, 7, and 14 days, and vasculogenesis was assessed by implantation into the chorio-allantoic membrane in chick embryos for 7 days. In vivo biocompatibility was assessed by subcutaneous implantation in rats for 1, 3, and 6 weeks. The membranes consisted of random fibers of PLLA-PU with fiber diameters of 0.47 and 0.12 µm, respectively. Significantly higher cell proliferation and migration of MC3T3 cells at 3, 7, and 14 days were shown on plasma-coated membranes compared with uncoated membranes. Further, it was found that plasma-coated membranes were more angiogenic than controls. In vivo implantation of membranes in rats did not reveal any gross toxicity to the materials, and wound healing was comparable with the native tissue repair (sham group). We therefore present a plasma-functionalized electrospun composite polymer membrane for use in the treatment of fistulas. These membranes are flexible, non-cytotoxic, and angiogenic, and we hope it should lead to permanent closure of oronasal fistula.

The changing regulatory landscape for biomedical implants and its relationship to withdrawal of some vaginal mesh products.

PURPOSE OF REVIEW: Advancements in biomedical engineering and advanced therapies including tissue engineering products necessitate revisions to the regulation and governance of their production and use to ensure patient safety. In this review, the current regulations and recent improvements on the governance of biomedical devices are reviewed. RECENT FINDINGS: Current regulations on approval of biomedical devices failed to address some important aspects related to the definition of biocompatibility of medical implants. The main issue was that the failure to establish design requirements for a specific application - in this case, the pelvic floor. Another issue was the lack of knowledge on disease mechanisms leading to an inability to define clear targets for surgical treatment. A clear example of this is the recent vaginal mesh scandal. SUMMARY: Surgical innovations are inherently challenging. It is no surprise that the regulatory landscape lags behind advancements in biomedical technologies. Very recent modifications to the available regulations particularly in Europe aim to establish a robust, transparent, predictable and sustainable regulatory framework for medical devices, which ensures patient safety while supporting innovation.

Improving the biocompatibility of biomaterial constructs and constructs delivering cells for the pelvic floor.

PURPOSE OF REVIEW: Interactions between biomaterials and biomaterial-delivering cells and the host tissues are complexly affected by the material itself, the ultrastructure of the overall construct and cells and other bioactive factors involved. The aim of this review is to review the current understanding on the definitions of biocompatibility and current advances in improving biocompatability of tissue-engineered constructs. RECENT FINDINGS: Some synthetic materials are associated with more foreign body reactions compared with natural materials; however, they allow fabrication of materials with a great diversity of physical and mechanical properties. Material design strategies can be tailored to mimic the natural extracellular matrix topography. There are also advancements in the pharmacological functionalization of materials with improved angiogenic potential that can lead to better tissue response. Stem cells are also used to improve the tissue response of tissue-engineered materials; however, the recent regulations on regenerative medicine products necessitate significant regulatory approval processes for these. SUMMARY: The biggest challenge faced in translation of tissue-engineered constructs into clinical practice relates to their engraftment and poor tissue integration into the challenging wound bed of the pelvic floor. Biocompatibility of tissue engineered constructs can theoretically be improved by the incorporation of bioactive agents, such as vitamins C or oestradiol.

Tissue engineering for the pelvic floor.

PURPOSE OF REVIEW: To set in context the challenge of developing tissue-engineered constructs for use in the female pelvic floor compared with at least 30 years of research progress in tissue engineering for other tissues. RECENT FINDINGS: The relative lack of information on the mechanical requirements of the pelvic floor in women who have suffered damage to these tissues is a major challenge to designing tissue-engineered materials for use in this area. A few groups are now using autologous cells and biomaterials to develop constructs for repair and regeneration of the pelvic floor. Progress with these has reached early stage evaluation in small animal models. Meanwhile the regulatory challenge of introducing laboratory-expanded cell therapy into the clinic is prompting groups to look at alternatives, such as using lipoaspirate retrieved in theatre as a source of adult stem cells for a number of tissues. In our group, we have begun to look at lipoaspirate for repair of the pelvic floor. SUMMARY: There is a need for research to harvest the advances made over the last 30 years in developing tissue-engineered constructs for several tissues to now tackle the problems of the weakened pelvic floor. At present, there are relatively few groups engaged in this challenge despite the growing clinical need.

Designing new synthetic materials for use in the pelvic floor: what is the problem with the existing polypropylene materials?

PURPOSE OF REVIEW: This review identifies the clinical complications associated with the design of the current polyproplylene mesh materials used for the treatment of stress urinary incontinence and pelvic organ prolapse. Following on from this, new alternative materials under development for pelvic floor repair are reviewed. RECENT FINDINGS: It is well accepted that the textile properties of the current polypropylene surgical meshes are not suitable for the pelvic floor environment. This together with the chemical nature of the current mesh leads to complications whenever implanted in the pelvic floor of women. New alternative materials for the repair of the pelvic floor have been developed with properties designed to be more appropriate for the biomechanical requirements and implantation requirements for the pelvic floor to reduce these clinical complications. To support this, these newer materials are being rigorously tested using more appropriate in-vitro regimes and animal models. SUMMARY: This chapter summarizes developments in the design of new materials for pelvic floor repair. These are being subjected to preclinical testing to exclude materials, which might fail to work in this dynamic environment by either showing a poor mechanical match to the requirements of the tissue or by provoking sustained inflammation. The hope is that new materials will prove effective without causing the high incidence of unacceptable side-effects currently seen with polypropylene mesh implants.

Use of a simple in vitro fatigue test to assess materials used in the surgical treatment of stress urinary incontinence and pelvic organ prolapse.

AIMS: Stress urinary incontinence and pelvic organ prolapse are very common conditions with a proportion of patients requiring implantation of synthetic materials for a durable repair. However increasing numbers of post-surgical complications have been reported related to the use of polypropylene meshes. One hypothesis for the adverse response is poor mechanical matching of the relatively stiff polypropylene mesh particularly as materials in the pelvic floor will need to cope with decades of distension as occurs with increase of intraabdominal pressure on coughing, laughing, or sneezing. METHODS: In this study we have undertaken a very simple fatigue testing regime to compare the mechanical abilities of six materials. Four commercial meshes in clinical use and two novel electrospun materials not yet evaluated in the clinic were assessed using a uniaxial tensile test. This was performed on six samples of each dry material and on another six samples of each material after just 3 days of fatigue conditions using a dynamic bioreactor. RESULTS: The four commercial materials showed permanent mechanical deformation after just 3 days of stretching these materials by 25% elongation on a regular dynamic cycle, whereas the two new materials presented more elastic properties without deformation. CONCLUSIONS: We suggest that a test as simple as this 3-day fatigue testing is sufficient to distinguish between materials which have already been found to cause complications clinically and newer materials yet to be tested clinically which will hopefully prove more mechanically appropriate for implantation in the pelvic floor.

Visualisation of the insertion of a membrane for the treatment of preterm rupture of fetal membranes using a synthetic model of a pregnant uterus.

Preterm premature rupture of fetal membranes is a leading cause of preterm delivery. Preterm labour can compromise fetal survival, and even if a pregnancy affected by preterm premature rupture of fetal membrane continues, major complications associated with leakage of amniotic fluid and risk of infection can affect the normal development and survival of the baby. There are limited management options for preterm premature rupture of fetal membrane other than delivery of the baby if ascending infection (chorioamnionitis) is suspected. We have previously reported the development and characterisation of an implantable membrane with the aim of using it to occlude the internal os of the cervix, in order to prevent amniotic fluid loss, allow fluid reaccumulation and reduce the risk of chorioamnionitis. For this, an electrospun biocompatible and distensible bilayer membrane was designed with mechanical properties similar to the human amniotic membrane. In this study, we consider the effects of sterilization on the membrane, how to insert the membrane and visualise it using routine clinical methods. To do this, we used e-beam sterilisation and examined the ability of the membrane to adhere to ex vivo human cervical tissues. We also studied its insertion into a custom-synthesised model of a 20-week pregnant uterus and imaged the membrane using ultrasound. Sterilisation produced minor effects on physical and mechanical properties, but these did not affect the capacity of the membrane to be sutured or to provide a fluid barrier. We demonstrated that fibrin glue can successfully adhere the bilayer membrane to cervical tissues. Finally, we demonstrated that the membrane can be inserted through the cervix as well as visualized in place using ultrasound imaging and an endoscope. In summary, we suggest this membrane is a candidate for further development in an appropriate animal model, supported by appropriate imaging, to precede possible future human studies if judged to demonstrate satisfactory safety and efficacy profiles.

Modulation of the Early Host Response to Electrospun Polylactic Acid Matrices by Mesenchymal Stem Cells from the Amniotic Fluid.

PURPOSE: The reconstruction of congenital diaphragmatic hernia or other congenital soft tissue defects often requires implants. These can be either degradable or permanent, each having their advantages. Whatever type is being used, the host response induced by implants plays a crucial role to determine the outcome. Macrophages are pivotal during implant remodeling; they are plastic and acquire in response to environmental stimuli either an inflammatory status and mediate subsequent fibrosis or a regulatory status and facilitate functional remodeling. Matrices engineered with mesenchymal stem cells (MSCs) have the capacity to modulate the host immune reaction. MSCs are believed to promote constructive remodeling of the implant through a regulatory macrophage response among others. Herein, we evaluate this potential of MSC derived from the amniotic fluid (AF-MSC), an interesting MSC type for neonatal reconstruction, on electrospun polylactic acid (PLA) scaffolds. METHODS: We seeded AF-MSC at a density of 1.105/cm2 on electrospun PLA matrices and determined cell viability. In vivo, we used cell-seeded or cell-free PLA matrices for subcutaneous implantation in immune competent rats. The host immune response was evaluated by histomorphometry at 14 days postoperatively. RESULTS: The PLA matrix supported adherence and proliferation of AF-MSC. Fourteen days after implantation, PLA matrices were well penetrated by inflammatory cells, new blood vessels, and collagen fibers. AF-MSC-seeded scaffolds were associated with a similar response yet with a decreased number of eosinophils, increased matrix degradation and collagen fiber deposition compared with controls. The amount of total macrophages and of M2-subtype was similar for all animals. CONCLUSION: Electrospun PLA matrices are a suitable substrate for short-term culture of AF-MSC. In rats, addition of AF-MSC to PLA matrices modulates the host response after subcutaneous implantation, yet without a difference in macrophage profile compared with control.

Developing Repair Materials for Stress Urinary Incontinence to Withstand Dynamic Distension.

BACKGROUND: Polypropylene mesh used as a mid-urethral sling is associated with severe clinical complications in a significant minority of patients. Current in vitro mechanical testing shows that polypropylene responds inadequately to mechanical distension and is also poor at supporting cell proliferation. AIMS AND OBJECTIVES: Our objective therefore is to produce materials with more appropriate mechanical properties for use as a sling material but which can also support cell integration. METHODS: Scaffolds of two polyurethanes (PU), poly-L-lactic acid (PLA) and co-polymers of the two were produced by electrospinning. Mechanical properties of materials were assessed and compared to polypropylene. The interaction of adipose derived stem cells (ADSC) with the scaffolds was also assessed. Uniaxial tensiometry of scaffolds was performed before and after seven days of cyclical distension. Cell penetration (using DAPI and a fluorescent red cell tracker dye), viability (AlamarBlue assay) and total collagen production (Sirius red assay) were measured for ADSC cultured on scaffolds. RESULTS: Polypropylene was stronger than polyurethanes and PLA. However, polypropylene mesh deformed plastically after 7 days of sustained cyclical distention, while polyurethanes maintained their elasticity. Scaffolds of PU containing PLA were weaker and stiffer than PU or polypropylene but were significantly better than PU scaffolds alone at supporting ADSC. CONCLUSIONS: Therefore, prolonged mechanical distension in vitro causes polypropylene to fail. Materials with more appropriate mechanical properties for use as sling materials can be produced using PU. Combining PLA with PU greatly improves interaction of cells with this material.

Evaluating Alternative Materials for the Treatment of Stress Urinary Incontinence and Pelvic Organ Prolapse: A Comparison of the In Vivo Response to Meshes Implanted in Rabbits.

PURPOSE: Serious complications can develop with the mesh implants used for stress urinary incontinence and pelvic organ prolapse surgery. We evaluated 2 materials currently in clinical use and 2 alternative materials using a rabbit abdominal model to assess host response and biomechanical properties of the materials before and after implantation. MATERIALS AND METHODS: Poly-L-lactic acid and polyurethane meshes were electrospun to be compared to commercially available polypropylene and polyvinylidene fluoride meshes. A total of 40 immunocompetent full-thickness abdominal wall defect rabbit models were used, including 8 in each of the poly-L-lactic acid, polyurethane, polyvinylidene fluoride and polypropylene experimental groups, and sham controls. Two 20 mm defects were created per animal and primarily repaired. The experimental groups then underwent onlay of each repair material while sham controls did not. Four rabbits per group were sacrificed at days 30 and 90. Abdominal wall specimens containing the defect with or without repair material were explanted to be assessed by histology (hematoxylin and eosin staining, and immunohistochemistry) and biomechanical testing at 30 and 90 days. RESULTS: At 90 days of implantation tissues repaired with all 4 materials showed biomechanical properties without significant differences. However, polypropylene and polyvinylidene fluoride meshes demonstrated a sustained chronic inflammatory response profile by 90 days. In contrast, poly-L-lactic acid and polyurethane meshes integrated well into host tissues with a decreased inflammatory response, indicative of constructive remodeling. CONCLUSIONS: Poly-L-lactic acid and polyurethane alternative materials achieved better host integration in rabbit models than current synthetic repair materials.

Biodegradable scaffolds designed to mimic fascia-like properties for the treatment of pelvic organ prolapse and stress urinary incontinence.

There is an urgent clinical need for better synthetic materials to be used in surgical support of the pelvic floor. The aim of the current study was to construct biodegradable synthetic scaffolds that mimic the three-dimensional architecture of human fascia, which can integrate better into host tissues both mechanically and biologically. Therefore, four different polylactic acid (PLA) scaffolds with various degrees of fibre alignment were electrospun by modifying the electrospinning parameters. Physical and mechanical properties were assessed using a BOSE electroforce tensiometer. The attachment, viability and extracellular matrix production of adipose-derived stem cells cultured on the polylactic acid scaffolds were evaluated. The bulk density of the scaffolds decreased as the proportion of aligned fibres increased. Scaffolds became stronger and stiffer with increasing amounts of aligned fibres as measured along the axis parallel to the fibre alignment. In addition, more total collagen was produced on scaffolds with aligned fibres and was organised in the direction of the aligned fibres. In conclusion, the electrospinning technique can be easily modified to develop biodegradable scaffolds with a spectrum of mechanical properties allowing extracellular matrix organisation towards human-like fascia.

Development of an implantable synthetic membrane for the treatment of preterm premature rupture of fetal membranes.

Preterm premature rupture of fetal membranes is a very common condition leading to premature labour of a non viable fetus. Significant morbidities may occur when preterm premature rupture of fetal membranes management is attempted to prolong the pregnancy for fetal maturation. Reducing the rate of loss of amniotic fluid and providing a barrier to bacterial entry may allow the pregnancy to continue to term, avoiding complications. Our aim is to develop a synthetic biocompatible membrane to form a distensible barrier for cervical closure which acts to reduce fluid loss and provide a surface for epithelial ingrowth to help repair the damaged membranes. Therefore, a bilayer membrane was developed using an electrospinning technique of combining two FDA-approved polymers, poly-L-lactic acid (PLA) and polyurethane (Z3) polymer. This was compared to a plain electrospun Z3 membrane. The physical and mechanical properties were assessed using scanning electron microscope images and a BOSE tensiometer, respectively, and compared to native fetal membranes. The performance of the membranes in preventing fluid loss was assessed by measuring their ability to support a column of water. Finally the ability of the membranes to support cell ingrowth was assessed by culturing adipose-derived stem cells on the membranes for two weeks and assessing metabolic activity after 7 and 14 days. The physical properties of the bilayer were similar to that of the native fetal membranes and it was resistant to fluid penetration. This bilayer membrane presented mechanical properties close to those for fetal membranes and showed elastic distention, which may be crucial for progress of the pregnancy. The membrane was also able to retain surgical sutures. In addition, it also supported the attachment and growth of adipose-derived stem cells for two weeks. In conclusion, this membrane may prove a useful approach in the treatment of preterm premature rupture of fetal membranes and now merits further investigation.