Adipose derived stromal cells cultured on poly(trimethylene carbonate) based biomaterials for pelvic organ prolapse applications

Abstract

Pelvic organ prolapse (POP) is common problem affecting large number of women worldwide. In POP, muscles and connective tissues keeping the structure and support in pelvic floor are weakened, leading to descent of pelvic organs. Collagen is the main component in the connective tissue, thus changes in collagen composition leads to decreased strength in pelvic floor. Pregnancy, vaginal delivery and aging are the most common risk factors changing the strength of muscular and connective tissue in pelvic floor. Estimated lifetime risk of women ending up for a surgery due to POP is 11-13 %. Yet, recurrence rate after primary surgery using patient’s own tissue is even 30 %. Non-absorbable synthetic polypropylene meshes have been commonly used as repair materials to prevent recurrence of POP. However, those meshes have been withdrawn from the market due to mesh-related complications including mesh erosion and pelvic pain. Thus, development of new and safe repair material for POP surgery is essential. Tissue engineering, utilizing cells, biodegradable biomaterials and active agents, could be one potential approach for future POP surgery. Ideal biomaterial should support the prolapsed tissue by withstanding the forces in pelvic floor. In addition, it should interact with the surrounding tissue and cells and promote important cellular functions. Poly(trimethylene carbonate) (PTMC) is flexible and biodegradable synthetic elastomer. Biocompatibility, highly tunable mechanical properties and unique degradation of PTMC has increased its potential in various soft tissue engineering applications. Active agents can be embedded into biomaterial to promote interaction with cells. One interesting active agent is ascorbic acid and its derivative L-ascorbic acid 2-phosphate (A2P), as they are shown to increase the proliferation and collagen production of cells. In this study, PTMC membranes embedded with 0 wt%, 5 wt% and 10 wt% of A2P were cultured with adipose derived stromal cells (ASCs) to assess the effect of A2P to cellular response at time points d1, d7 and d14. Scanning electron microscope imaging was used to assess cell morphology. Viability of ASCs was determined with Live/Dead® assay. Cell proliferation and collagen production was analyzed with CyQUANT® and Sircol™ assays. Expression of collagen I, vinculin and αSMA was visualized with immunocytochemistry, and gene expression of collagen I, collagen III, elastin and αSMA was measured with quantitative real-time polymerase chain reaction. In addition to cell culture studies, the hydrophilicity of PTMC membranes was determined by contact angle measurements. Incorporation of A2P increased the hydrophilicity of PTMC membrane and altered the surface morphology making it more favorable for cells to attach, as ASCs seemed to spread evenly and form dense cells sheets on membranes containing A2P. ASCs remained viable in all materials and hardly any dead cells were visible. However, the cell amount on plain PTMC membrane was extremely low. Embedded A2P increased the proliferation and collagen production of ASCs. Immunostaining showed that produced collagen remained mostly inside the cells. Furhtermore, increased expression of αSMA indicate potential myofibroblastic differentiation. PTMC membrane containing 5 wt% of A2P have the best outcomes on cell proliferation, collagen production and differentiation. As conclusion, PTMC membranes embedded with A2P could be potential materials for POP applications.