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

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

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