Development of an engineered tissue designed for pelvic floor repair

Stress urinary incontinence (SUI) and pelvic organ prolapse (POP) are diseases related to weakness of supportive tissues of the pelvic floor due to altered collagen production in middle-aged women and traumatic processes in younger women such as pregnancy and vaginal delivery. Currently there is no recommended material for use in the surgical management of these disorders. Synthetic non-absorbable materials, such as polypropylene mesh produce a vigorous inflammatory response followed by dense fibrosis and have been associated with serious complications such as exposure. By contrast acellular biological materials have a tendency toward rapid absorption with questionable long-term mechanical integrity and concerns regarding early failure. Our approach aims to develop a tissue engineered repair material (TERM) to provide the long-term durability of synthetic non-absorbable materials whilst avoiding complications such as exposures and pain. The TERM is composed of a scaffold designed to degrade slowly whilst the inclusion of autologous cells is anticipated to produce a new extracellular matrix (ECM) to remodel fascial tissue for long-term restoration of the mechanical properties. Biodegradable poly-(L)-lactic acid (PLA) scaffolds were identified as the candidate material being more cell compatible in vitro than materials currently used to treat SUI and POP, and with mechanical properties close to the range of native tissues of the pelvic floor. A comparison of oral fibroblasts and adipose-derived stem cells (ADSCs) showed similar results when these cells were cultured on PLA scaffolds to develop a TERM in terms of metabolic activity, ECM production and mechanical properties. Of the two, ADSCs were chosen for further experiments since these cells have been shown in the literature to have regenerative potential and also to be immunosuppressive and to stimulate angiogenesis. The number of cells seeded on the scaffolds, the period of culture and culture conditions were optimized for the production of the best TERM candidate. On the other hand, no significant effects were found when exploring chemical and mechanical stimulation with the aim of increasing ECM production. The host response against the PLA scaffolds implanted cell-free and with ADSCs was studied in rats. The acute host response showed that after an inflammatory response, new collagen ingrowth and blood vessels were developed in all samples. Work was then focussed on the modification of the electrospinning rig to develop a variety of PLA scaffolds with different mechanical properties due to different fibre configuration. Finally, the potential of ADSCs to develop the TERM was assessed using cells from different donors, as well as examining whether this potential was preserved when these cells were rapidly isolated from fat using an enclosed system. In summary, we identified a suitable candidate material, cell candidate and culture conditions to develop a TERM designed for pelvic floor repair. Then, an initial animal study suggested a host response against our TERM leading to constructive remodelling for integration into the native tissues. Finally, a range of PLA scaffolds were produced with improved mechanical properties and preliminary data showed the potential to rapidly isolate ADSCs which were used to develop a TERM in vitro

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

Production of ascorbic acid releasing biomaterials for pelvic floor repair

Objective: An underlying abnormality in collagen turnover is implied in the occurrence of complications and recurrences after mesh augmented pelvic floor repair surgeries. Ascorbic acid is a potent stimulant of collagen synthesis. The aim of this study is to produce ascorbic acid releasing poly-lactic acid (PLA) scaffolds and evaluate them for their effects on extracellular matrix production and the strength of the materials. Materials and methods: Scaffolds which contained either L-ascorbic acid (AA) and Ascorbate-2-Phosphate (A2P) were produced with emulsion electrospinning. The release of both drugs was measured by UV spectrophotometry. Human dermal fibroblasts were seeded on scaffolds and cultured for 2 weeks. Cell attachment, viability and total collagen production were evaluated as well as mechanical properties. Results: No significant differences were observed between AA, A2P, Vehicle and PLA scaffolds in terms of fibre diameter and pore size. The encapsulation efficiency and successful release of both AA and A2P were demonstrated. Both AA and A2P containing scaffolds were significantly more hydrophilic and stronger in both dry and wet states compared to PLA scaffolds. Fibroblasts produced more collagen on scaffolds containing either AA or A2P compared to cells grown on control scaffolds. Conclusion: This study is the first to directly compare the two ascorbic acid derivatives in a tissue engineered scaffold and shows that both AA and A2P releasing electrospun PLA scaffolds increased collagen production of fibroblasts to similar extents but AA scaffolds seemed to be more hydrophilic and stronger compared to A2P scaffolds

Biomimetic poly(glycerol sebacate)/poly(L-lactic acid) blend scaffolds for adipose tissue engineering

Large three-dimensional poly(glycerol sebacate) (PGS)/poly(l-lactic acid) (PLLA) scaffolds with similar bulk mechanical properties to native low and high stress adapted adipose tissue were fabricated via a freeze-drying and a subsequent curing process. PGS/PLLA scaffolds containing 73 vol.% PGS were prepared using two different organic solvents, resulting in highly interconnected open-pore structures with porosities and pore sizes in the range of 91–92% and 109–141 μm, respectively. Scanning electron microscopic analysis indicated that the scaffolds featured different microstructure characteristics, depending on the organic solvent in use. The PGS/PLLA scaffolds had a tensile Young’s modulus of 0.030 MPa, tensile strength of 0.007 MPa, elongation at the maximum stress of 25% and full shape recovery capability upon release of the compressive load. In vitro degradation tests presented mass losses of 11–16% and 54–55% without and with the presence of lipase enzyme in 31 days, respectively. In vitro cell tests exhibited clear evidence that the PGS/PLLA scaffolds prepared with 1,4-dioxane as the solvent are suitable for culture of adipose derived stem cells. Compared to pristine PLLA scaffolds prepared with the same procedure, these scaffolds provided favourable porous microstructures, good hydrophilic characteristics, and appropriate mechanical properties for soft tissue applications, as well as enhanced scaffold cell penetration and tissue in-growth characteristics. This work demonstrates that the PGS/PLLA scaffolds have potential for applications in adipose tissue engineering

Developing repair materials for stress urinary incontinence to withstand dynamic distension

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.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.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.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.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

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.status: publishe

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.status: accepte

Evaluating alternative materials for the treatment of stress urinary incontinence and pelvic organ prolapse -a comparison of the in vivo response to meshes of polypropylene, polyvinylidene fluoride, poly-lactic acid and polyurethane implanted in rabbits for 3 months

Serious complications can occur with the mesh implants used for stress urinary incontinence and pelvic organ prolapse surgery. We aimed to evaluate two materials, currently in clinical use and two alternative materials using a rabbit abdominal model to assess host response and biomechanical properties of the materials pre and post implantation.status: publishe

Collagen production of cells cultured on scaffolds.

<p>This was measured by Sirius red assay. Absorbance measured at 490nm per gram of scaffold, n = 3±SEM ***p<0.001.</p

Values for Mechanical properties before and after 7 days of dynamic loading.

<p>Young’s Modulus (<b>top</b>) and ultimate tensile strength (<b>bottom</b>) calculated from stress curves before and after 7 days of uniaxial distension (n-2±SEM), *p<0.05, **p<0.01, ***p<0.001. Dotted lines represent values of healthy paravaginal tissues [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149971#pone.0149971.ref024" target="_blank">24</a>].</p